The game begins as Zero is rescuing a kidnapped scientist from a warehouse. The scientist is found but immediately killed by V, a Russian mobster, who escapes. His mission failed, Zero visits his psychiatrist to receive his daily Chronos injection and a dossier on his next target, Josh Rose. Zero fights his way to Rose, who commits suicide. On his way home, Zero befriends a young girl living next door to his apartment in the Third District.
Next, Zero is sent to kill DJ Electrohead, a disc jockey involved in the drug trade, and is explicitly warned not to speak to him. Zero infiltrates his nightclub and can either kill Electrohead or listen to him confess to using Chronos before he is shot dead by V, who again flees. Zero's fourth target is Fa Yuan, a prison inmate. Upon arriving, Zero finds the prison staff massacred and his target already dead. SWAT officers arrive and swarm the cellblock as Zero escapes. At his apartment, the girl gives him a videotape she claims V left for him, containing a recording of V torturing and killing Zero's other neighbors.
Zero finds his psychiatrist's office closed the next morning and is picked up by V in a limousine on his way back. V offers to partner with Zero, who flatly refuses and is tossed out of the limo. He tracks down V to a movie backlot and fights through various film sets to reach him. Their fight is interrupted by Snow, a swordswoman working with V, who threatens Zero and leaves with V.
After experiencing hallucinations, Zero is assigned to kill Al-Qasim, a wealthy industrialist. Zero comes upon V and his men already storming Al-Qasim's mansion and is captured. When Zero uses precognition to mock him, V shoots Al-Qasim and leaves Zero to be killed by his henchman, Mr. Kissyface. Zero kills Kissyface and chases V and his men down the highway on a motorcycle. Before Zero can kill him, V is dismembered and then abducted by another swordsman, who displays similar abilities to Zero's. Before Zero flees, the swordsman identifies himself as 'the Dragon.'
Zero meets V again in another hallucination, who reveals his visions are the result of Chronos withdrawal—without it, he will be trapped in time, unable to die. Zero's psychiatrist sends him to a Chinatown casino to hunt down The Dragon. Within the Casino, Zero encounters The Dragon, as well as Headhunter, another Chronos user. Zero duels Headhunter while The Dragon flees the scene; the duel ends in a draw, and Headhunter escapes. While leaving, Zero acquires a tape containing the Prison's security footage from the night of Fa Yuan's murder. The tape footage shows The Dragon storming the prison and questioning Fa Yuan, who mentions Leon von Alvensleben, the creator of Chronos.
After finding the psychiatric office closed, Zero is directed to a slaughterhouse by an unknown caller. The slaughterhouse proves to be a front for a training facility for 'NULLs', Chronos-enhanced super soldiers used by New Mecca in the Cromag war. While progressing through the facility, Zero is addressed by Alvensleben, who observes and speaks to him through a series of monitors throughout the facility.
After determining Zero was once an elite Gamma NULL, Alvensleben reveals the project was a failure, and its subjects marked for death. Realizing that the footage of the scientist is a recording made years ago, Zero thaws the real Alvensleben from cryostasis, killing him.
Zero locates and confronts the Psychiatrist, who confirms that Zero was a NULL soldier, and that his targets have all been related to Chronos. He then sends Zero to destroy the contents of Al-Qasim's safe room in an underground bunker. At the bottom, he finds Headhunter guarding the vault. Headhunter confirms that she is also a NULL, and that she is suffering Chronos withdrawal after Al-Qasim's death. Zero kills her and enters the vault, where he finds a woman and children cowering in fear. Unable to proceed, he flees.
Zero discovers his psychiatrist preparing to flee the city, brutally beats him to death, and injects himself with a massive dose of Chronos. Alternatively, if Zero interrupts the Psychiatrist constantly during conversations and deliberately disobeys his directives during missions, the Psychiatrist becomes enraged and injects himself with an unknown drug, transforming into a monstrosity. Zero duels the monstrosity and kills him. Upon Zero's victory, the scene immediately cuts to him calmly seated in the office, the Psychiatrist's human self dead by a stab to the head rather than by being beaten to death. Zero searches the Psychiatrist's belongings and leaves without visibly injecting himself with Chronos.
Returning to his apartment, he finds it burglarized, the young girl gone, and a cryptic note left behind. The landlady states that no children lived in the building. When questioned, Zero runs, and the police give chase.
Zero's recurring nightmare is replayed, minus the distortion. Zero is revealed to have been the NULL soldier responsible for shooting the Scientist, rather than the child who hid. Having completed his objective, Zero exits the scene of the nightmare and walks over to stand next to The Dragon, who is shown to have been Zero's comrade in arms during the Cromag War.
In a mid-credits scene, Snow informs her superior of V's death at the hands of The Dragon, while The Dragon contemplates a board of evidence at a safe house. In a post-credits scene, Comedy and Tragedy taunt the little girl as she cowers in fear.
