In Tokyo, Yuichi Kimura seeks revenge after his son Wataru is pushed off a rooftop, boarding a bullet train in search of the attacker. Guided by his handler Maria Beetle, operative "Ladybug" is assigned to retrieve a briefcase from the same train after the previous contract, Carver, calls in sick. Ladybug is reluctant, as his recent string of bad luck during his jobs resulted in accidental deaths. Also on the train is a young woman disguised as a schoolgirl, codenamed "The Prince", and two English assassin brothers codenamed "Lemon" and "Tangerine", who are assigned to escort both the briefcase and "The Son", the son of Russian-born Yakuza boss "The White Death".

Ladybug discreetly steals the briefcase, but is attacked by another assassin, "The Wolf", who mistakenly blames Ladybug for fatally poisoning his entire wedding party. After a brief fight with a confused Ladybug, the Wolf accidentally kills himself. Ladybug arranges the Wolf's corpse to look like a sleeping passenger. Meanwhile, the Prince reveals to Yuichi that she pushed Wataru off the roof to lure him to the bullet train as part of a plan to have him kill the White Death, and that she has a henchman holding Wataru hostage in the hospital to ensure that Yuichi follows her orders. Meanwhile, the Son is poisoned and dies in the same manner as the Wolf's wedding party.

Ladybug offers the briefcase to Lemon in return for getting off the bullet train. Lemon suspects that Ladybug killed the Son, leading to a fight in which Lemon is knocked unconscious after realising Ladybug was innocent. The Prince and Yuichi find the briefcase and booby-trap it with explosives, and rigs his gun to explode if fired. Ladybug encounters Tangerine and kicks him off the bullet train after another scuffle, but he jumps and climbs back aboard from outside. A suspicious Lemon shoots Yuichi, but collapses after drinking from a water bottle Ladybug had previously spiked. The Prince shoots Lemon and conceals him and Yuichi in a bathroom. Ladybug encounters yet another assassin, "The Hornet", who poisoned the Son and the Wolf's wedding party with boomslang venom. After a struggle, both are exposed to the venom, and Ladybug steals her antivenom to save himself.

Tangerine runs into the Prince, realizing that she shot Lemon, only to be interrupted by Ladybug and killed before he can shoot her. At the next stop, Yuichi's father, "The Elder", boards the bullet train. He recognizes the Prince, and informs her that Wataru is safe. After she flees, the Elder tells Ladybug he will remain to confront the White Death, who killed his wife while taking over the Yakuza. Finding an injured Yuichi and Lemon still alive, the four work together to face the White Death. At Kyoto, Ladybug gives the White Death the briefcase. The Prince, revealed to be the White Death's daughter, tries to goad him into shooting her with the rigged gun, but fails.

The White Death explains that everyone on the bullet train was linked to death of his wife, and that he hired them hoping they would kill each other. Only Ladybug was filling in for Carver, the main target of the White Death. The White Death's henchmen open the briefcase, which explodes, knocking Ladybug and the White Death back onto the bullet train. The White Death's remaining henchmen board and battle the assassins, while the Elder duels the White Death, causing the bullet train to crash into downtown Kyoto. Emerging from the wreck and impaled with the Elder's katana, the White Death