NOTE: This spoiler was submitted by Jeremy

The film opens at a hospital in Tokyo. Yuichi Kimura/”The Father” (Andrew Koji) is by the bedside of his son Wataru (Kevin Akiyoshi Ching), who was pushed off a rooftop while his father was absent. Yuichi’s father, “The Elder” (Hiroyuki Sanada), arrives and reminds his son that fathers are supposed to protect their families. Yuichi goes off to find whoever hurt his son.

An assassin named Ladybug (Brad Pitt) is returning to work after a leave of absence. He considers the name ironic, as he doesn’t believe he is lucky like ladybugs usually are. He is also filling in for another assassin, Carver, who is sick. Ladybug speaks to his handler, Maria Beetle (Sandra Bullock), who assigns him to board a bullet train heading from Tokyo to Kyoto. His task is to recover a briefcase containing money with a train sticker on the handle. As he makes his way over, Ladybug gets bumped into by Yuichi, causing him to drop a key to a locker and his train ticket. Ladybug breaks into the locker to find a gun in there, but he declines to take it, despite Maria’s insistence.

Onboard the train are “twin” brother assassins, Tangerine (Aaron Taylor-Johnson) and Lemon (Brian Tyree Henry). They were sent by a crime boss known as “The White Death” (Michael Shannon) to recover his son (Logan Lerman), as he is a junkie that can’t be trusted on his own. Lemon lives much of his philosophy based off “Thomas The Tank Engine”, and he relates certain people to characters from the show. Tangerine explains to Lemon about how White Death was a Russian wanderer who ended up working as the right hand man of a Yakuza boss named Minegishi (Nobuaki Shimamoto), until he ended up forming his own gang and betraying Minegishi and killing him. While keeping the Son company, the brothers argue about how many people they killed altogether (one was an innocent bystander in the wrong place at the wrong time).

Yuichi boards the train after finding a lead on who pushed Wataru. He is tased by an assassin called “The Prince” (Joey King), who poses as an innocent schoolgirl to throw people off to her true nature. When he wakes up, she tells him that she pushed Wataru to lure Yuichi to her, as it was he who supplied Lemon and Tangerine with the briefcase. She wants Yuichi’s help in rigging the briefcase and giving it to White Death to kill him. Prince has also rigged a gun to blow his head up when the trigger is pulled. In order to force Yuichi to cooperate, Prince tells him that she has a henchman at the hospital ready to kill Wataru if he doesn’t hear from her.

Ladybug recovers the briefcase but is told by the conductor (Masi Oka) that he has to get off at the first stop because he didn’t have his ticket on him. Before he steps out, he is found by another assassin known as “The Wolf” (Bad Bunny). A flashback shows Wolf working his way up in the cartel under a boss called El Saguaro (Julio Gabay) and then finding a wife (Andrea Munoz). However, everyone at Wolf’s wedding was poisoned, including El Saguaro and Wolf’s wife. He believes Ladybug is responsible for their deaths, and he attempts to kill him. The two fight in the bar car before Wolf’s knife bounces off the briefcase and plunges into his heart, killing him. Ladybug covers up Wolf’s wounds and calls Maria. She informs him that he was a cocktail waiter at Wolf’s wedding, and the tequila served there was spiked with poison from a boomslang snake, which was stolen from a zoo in Tokyo and is actually on the train.

Lemon and Tangerine discover the briefcase is missing, and the Son has also been poisoned to death. They cover his face up and clean the blood before going off to find Ladybug. Ladybug encounters Lemon in the quiet car and recognizes him from a job in Johannesburg. They fight, with Ladybug trying to disarm Lemon. Ladybug ultimately knocks Lemon out and spikes his water bottle with sleeping powder. Ladybug then switches clothing with another passenger (Channing Tatum) and talks to Maria again. She tells him that another assassin, “The Hornet” (Zazie Beetz), was hired by White Death and is on the train. Meanwhile, Tangerine has to get off at the next stop briefly to meet with White Death’s goons to assure them that the job is going according to plan.

Tangerine finds Ladybug and fights him, believing that Ladybug poisoned White Death’s son. Ladybug opens an emergency door and causes the two to nearly fly out of the train before pulling themselves back in. White Death contacts Tangerine to let him know that if they do not show up with the Son and the briefcase, he will kill everyone on the train. The two attempt to fool White Death’s men by posing as brothers and using a different briefcase, but Ladybug accidentally opens it and blows their cover, forcing them to run back onto the train. Ladybug then kicks Tangerine off the train before it starts moving again, but he manages to hop back on board before it pulls away too fast.

Lemon comes across Yuichi and Prince, where he quickly determines that both of them are not there for noble purposes. Lemon shoots Yuichi, but he knows not to trust Prince. Unfortunately, he drank the spiked water and ends up passing out. Prince shoots Lemon in the chest and stashes him and Yuichi in the bathroom.

Ladybug runs into Hornet, who was in disguise on the train as an anime character before swapping clothes with the concession girl (Karen Fukuhara). She stole the boomslang snake from the zoo and uses its venom to kill her victims, as the venom congeals the blood and makes the victims bleed from every orifice. Hornet poisoned everyone at Wolf’s wedding, as she was also there as a pastry chef. Ladybug fights Hornet, who is there to collect her payment. She tries to stick him with a syringe containing the venom, but he takes a light jab while sticking her with the rest of it. Hornet tries to administer the antivenom, but Ladybug takes it from her first and watches her crawl away to die.

Tangerine finds Lemon’s body in the bathroom and tries to go after his killer. He talks to White Death again, who says that he bought every last ticket to Kyoto so there would only be the remaining few on the train. Tangerine runs into Prince and is almost fooled by her until he sees that Lemon left a sticker of the character Diesel from “Thomas”, meaning he knew she was a villain. Before Tangerine can kill Prince, Ladybug finds him again and fights him, ending with Ladybug shooting Tangerine in the neck, leaving him to bleed out before he can warn Ladybug about Prince.

Ladybug tries to get Prince off the train at the next stop, but she deliberately keeps them on, fooling him with her innocent act. The Elder then boards the train since he overheard Prince on the phone and knew something was wrong on the train. He sits near Ladybug and Prince until Ladybug gets scared away when the boomslang snake comes and bites him. The Elder confronts Prince, who prepares as her henchman gets ready to kill Wataru. However, The Elder prepared for this and had his own assassin posing as a nurse, and she kills Prince’s guy before he can touch Wataru. Prince flees, and Ladybug returns. The Elder talks to him about how Ladybug thinks he is unlucky, and leaves him with the belief that everything happens for a reason. He also tells the story of how he was Minegishi’s original confidant before White Death betrayed them, and he has vowed revenge on White Death for the death of his wife.

Yuichi and Lemon then wake up, as Lemon was wearing a bulletproof vest. When he sees that Tangerine is dead, he becomes enraged and tries to go after Ladybug, but Yuichi and The Elder have them work together as they prepare to face White Death.

The train arrives in Kyoto as White Death prepares to board. Prince confronts him, and it is revealed that they are father and daughter. She wanted to get his attention since he favored his son, and she attempts to get him to kill himself with the rigged gun, but he simply walks away from trying to kill her. The others confront him outside, while White Death’s goons try to open the rigged briefcase. White Death then explains that everyone on that train was there because he orchestrated it – he has wanted revenge for the death of his wife (Johanna Watts), who was killed in a car accident that was an assassination attempt on him. Lemon and Tangerine killed White Death’s men on a job in Bolivia, and he supplied them with info on other assassins. He also did not care for his son, as his wife’s attempt to bail him out again from his crimes led to her death. Hornet was lured there because she poisoned the surgeon that could have saved his wife’s life. Finally, he believes Ladybug is Carver (Ryan Reynolds), who orchestrated the assassination attempt. He was betting on all the assassins to kill each other. The briefcase then explodes just as Lemon gets the train to start running. Ladybug runs back on and is chased by White Death and his goons.

The Elder battles White Death on the train while Ladybug and Lemon fight off the henchmen as the train speeds out of control. As they kill the henchmen, Ladybug attempts to hit the emergency brakes. Lemon ends up throwing himself and the last henchman out of the train as they pass over a bridge. Before White Death can kill The Elder, Ladybug hits the brakes, and Elder seizes an opportunity to impale his enemy close to death. The train then derails and crashes into a town.

The surviving men exit the train, and White Death attempts to execute Ladybug, but he grabbed the rigged gun and blows half his own head off. Prince then tries to finish off Ladybug and the Kimuras, but she gets run over by a tangerine truck. Maria then arrives in person to rescue Ladybug while he parts with the Kimuras. He has embraced a positive outlook on life and believes he really is lucky…until a telephone pole comes crashing down onto Maria’s car, forcing them to walk.

As the credits start, we see that Lemon survived the fall off the train and hijacked the tangerine truck so he could run over Prince and avenge Tangerine.

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A bullet train going from Tokyo to Kyoto is boarded by a number of assassins. Ladybug is there because his handler Maria Beetle assigned him to recover a briefcase. The briefcase was brought on by "twin" brother assassins Lemon and Tangerine, who were hired by Russian crime boss White Death to recover his junkie son. Unfortunately, the son is poisoned, and the brothers believe Ladybug did it. A young schoolgirl assassin called The Prince lured Yakuza member Yuichi Kimura/The Father to help her kill White Death by pushing Yuichi's son off a roof and leaving him in a coma. Another assassin, The Wolf, tries to kill Ladybug after believing he was responsible for poisoning his wife and cartel boss at his wedding, while one final assassin, The Hornet, has stolen a deadly snake to use its poison on her victims.

Wolf accidentally kills himself fighting Ladybug when his knife bounces off the briefcase and into his heart. Hornet tries to poison Ladybug, but he poisons her too and steals her antivenom before she can use it, leaving her to die. Tangerine is killed in a gunfight with Ladybug. Yuichi is joined by his father, The Elder, who has Prince's henchman killed before he can touch his grandson. Ladybug, Lemon, Yuichi, and The Elder are left to work together to confront White Death.

When the train arrives in Kyoto, many revelations are made. First, Prince is White Death's daughter who wanted to kill him for not giving her the same attention he gave his son. Next, White Death arranged for all the assassins to be there as a result of his wife's death, which was part of a failed assassination attempt on him. He lured Lemon and Tangerine there because they killed his men in Bolivia, and was counting on the son to die because he blames him for causing his wife's death for bailing him out again. Hornet poisoned the surgeon that would have saved White Death's wife, and he believes that Ladybug is the assassin that orchestrated the hit, even though it was the guy Ladybug was filling in for on the job.

The showdown takes place as Lemon causes the train to go too fast. The Elder battles White Death, as he was responsible for the death of his wife after White Death seized power from The Elder's former boss. Ladybug and Lemon kill White Death's men before Lemon jumps out of the train with the last henchman. The train derails and crashes into a nearby town. White Death tries to kill Ladybug but gets his head blown up because he grabbed a gun rigged by Prince to explode when the trigger is pulled. Prince is also killed by Lemon, who survived his fall and hijacked a tangerine truck to avenge his brother. Maria then shows up to rescue Ladybug, while the Kimuras return home.