In modern-day Tokyo, grieving father Yuichi Kimura seeks revenge after an unknown assailant pushes his son off a rooftop, and has told him to meet them on a bullet train later in the night.

Meanwhile, former assassin "Ladybug", after recently graduating from therapy, is assigned to retrieve a briefcase from the same bullet train bound for Kyoto after the previous contact, Carver, calls in sick from a stomach flu. Ladybug is initially wary, as his recent string of bad luck during his jobs resulted in accidental deaths.

Also on the train is a young woman codenamed the "Prince" who attacked Yuichi's son, Yuichi, and two assassin brothers called "Lemon" and "Tangerine" who are assigned to escort both the briefcase and the son of a Russian-born Yakuza boss known as the "White Death", who have been hired by the "White Death" due to their roles in a job at Bolivia.

Ladybug steals the briefcase from Lemon and Tangerine but is attacked by another assassin called the "Wolf", who blames Ladybug for fatally poisoning his entire wedding party including his newlywed wife. After a brief fight, The Wolf's knife throw backfires and results in his death. Distraught, Ladybug stashes the briefcase away.

Meanwhile, the Prince reveals to Yuichi that she pushed his son off the roof to lure him to the train as part of an elaborate plan to assassinate the White Death, as well as the fact that she has a henchman holding his son hostage in the hospital, with orders to kill him should anything happen to her.

While Lemon and Tangerine search for the missing briefcase, the White Death's son is murdered in the same way as the Wolf's wedding guests. Ladybug offers the briefcase to Lemon in return for getting off the train. Lemon suspects that Ladybug killed the White Death's son, leading to a fight in which Lemon is knocked unconscious.

The Prince and Yuichi find the briefcase and booby-trap it with explosives to kill the White Death as well as a rigged gun as a second precaution. Ladybug encounters Tangerine and kicks him off the train, but Tangerine manages to climb back aboard. A suspicious Lemon shoots and injures Yuichi, but collapses after drinking from a water bottle Ladybug had earlier spiked with a sleeping drug.

The Prince shoots Lemon and stashes him and Yuichi in a bathroom. Ladybug encounters yet another assassin - the "Hornet", who poisoned the White Death's son and the Wolf's wedding party with boomslang venom. After a struggle during which they are both exposed to the venom, Ladybug steals her antivenom to save himself, leaving her to die.

Tangerine runs into the Prince and realizes that she shot Lemon, but Ladybug attacks before he can shoot her, killing Tangerine with his gun. Believing the Prince to be innocent, Ladybug agrees to protect her. Yuichi's father, the "Elder", boards the train and sees that the Prince is lying and informs her that Yuichi's son is safe (having appointed an undercover bodyguard for him who managed to kill The Prince's assassin).

After she flees, the Elder tells Ladybug that he is seeking revenge against the White Death, who killed his wife while taking over his Yakuza clan. They discover that Yuichi and Lemon are still alive, and the four work together to face the White Death.

At Kyoto, Ladybug gives the briefcase to the White Death. The Prince, revealed to be the White Death's estranged daughter, tries to goad him into shooting her with Yuichi's rigged gun, but she fails. The White Death explains that every assassin on the train, as well as his son, were responsible in some way for the death of his wife (except for the Wolf and Ladybug, who was filling in for another operative) and that he hired them all in the hopes they would kill each other.

The White Death's henchmen open the booby-trapped briefcase, which explodes, knocking Ladybug back onto the train. The White Death and his remaining henchmen board and battle the assassins. Their fight causes the train to hurtle out of control and crash into downtown Kyoto.

Emerging from the wreck, the White Death (who is fatally wounded by the Elder) tries to shoot Ladybug but is killed by the rigged gun. The Prince threatens Ladybug, Yuichi, and the Elder with a machine gun, but is suddenly struck and killed by a passing truck - later revealed to have been driven by Lemon, avenging Tangerine’s death in the process. Ladybug's handler Maria arrives to retrieve him as he celebrates finally getting off the bullet train.