Leon S. Kennedy stops as the Mizoil Gas Station to fill up his car, and sees a Zombie confronting a girl. The two run to his car, where she introduces herself as Claire Redfield. When they hit a block in the road deep in Raccoon City, the car is attacked by Zombies with an out-of-control truck coming at them. The two manage to get out of the car before it explodes, and are separated. Leon gets to the Raccoon Police Station second, and meets Claire just after a helicopter collides with the building. Hiding in a store room, he takes the time to get dressed into his uniform, and when he finally enters the station Claire is nowhere to be found. Once in the main hall, he finds a laptop that shows a massive man clad in black clothes noticing and destroying the camera. Searching in the S.T.A.R.S. Office, he finds a note left from her saying she has indeed already left. Leaving the room, he is discovered by T-00, a T-103 Tyrant sent to eliminate all police survivors. Leon is able to escape into an underground passage after collecting three medallions for a statue.
After fighting G1 Birkin, Leon meets Ada Wong after dropping a ladder for him. She claims to be an FBI agent, but refuses to work with him and locks him out of the prison cells. After restoring power to the basement, Leon follows and meets Ben Bertolucci, who has information of Police Chief Brian Irons' corruption, and offers a pass to get out of the parking garage if Leon lets him out. Leon refuses to trust a prisoner, and Ben is pulled into a wall by T-00 and his skull smashed. Ada walks in right after, and reveals he was an informant she was to meet. To gain access to Ben's cell, Leon explores the police station for two power panel parts. When fitted, he recovers the cardkey as well as an audio tape of an interview with Annette Birkin where he tried to get information on the G-Virus and its connection to the Raccoon City Orphanage. After saving Leon from the T-00 by crashing a SWAT van into him, Ada takes the tape and they head out into the streets.
Moving through the streets the two are forced off the main road by excavation work, and they travel through the Kendo Gun Shop. There, Leon is held up by Robert Kendo, owner of the shop. He becomes angry at their respective attitudes to his sick daughter, Emma, and they head back into his house behind the gunshop. The two then travel down into sewer tunnels on the other side of the excavated road. Moving under the city, Ada explains to Leon her mission for the FBI: to arrest Dr. Annette Birkin, who she holds responsible for spreading the virus around the city, but they must first find her secret lab, the location of which is secret due to manipulation of the civil authorities. Leon moves onto a lower level of the sewer tunnel, where he is chased down by a giant mutant Alligator, which eventually bites into a fuel tank which, when Leon shoots it, blows its head off.
Rejoined with Ada, they enter a passage with Dr. Birkin in as she investigates a body. When she realises Ada is there for the G-Virus, she shoots Leon; sets the body alight and locks herself behind a door. Ada searches through the sewers on her own, activating power to the facility through the use of a device that emits electromagnetic radiation. Pursued for a time by T-00, Ada finds an ID Wristband to let her into the NEST. She is trapped in an incinerator by Dr. Birkin, but is able to escape by using her device to force the hatch open. Following Dr. Birkin to a platform, Ada accuses her of murdering her husband William to get the G-Virus, which she denies. Ada is then forced off the platform, and falls to the ground with debris cut into her leg.
Leon wakes up and spots Ada in the Garbage Room through the window, and is forced to recover chess piece-shaped plugs to open the door, finding Claire already left on a cable car and returned it. When he does so, he is forced to fight G2, which he forces off a platform. Finding Ada, he pulls out the debris and sterilises the wound. She warns him to leave and save himself, but he won't leave her behind now, and the two head to NEST together. As they head in, Ada reminds him he isn't working in an official capacity with the FBI, and she'd rather he leave when she gets the G-Virus. He is unwilling to leave before everything is done, and they kiss. With Ada's leg still hurting, she gives him the ID Wristband to search the lab ahead of her.
Leon searches through the lab, but is forced to destroy a Monster Plant to get the necessary ID Wristband access to the lab, and runs into T-00 in the search. When he obtains a G sample from the lab, the self-destruct system activates - a system designed to deny intruders access to the virus. He is confronted by G2. Annette arrives and fires an anti-B.O.W. weapon at it, and reveals it is actually her husband, William, who infected himself with the G-Virus when Umbrella tried to steal it. Though he accepts her claim the viral outbreak wasn't intended, he derides her for trying to pass off responsibility onto Umbrella when as an administrator of NEST she was just as guilty. William begins to mutate into his G3 form and seriously injures Annette, and Leon is forced to fight him alone. When he succeeds in defeating William, he returns to Annette, who pleads to him that he destroy "G". Leon refuses, and insists it be taken to the FBI as evidence. Annette warns him not to trust Ada, believing her to be a mercenary sent to obtain "G" and sell it on the black market. When Leon meets up with Ada at the Administrator elevator, he asks her about it. Ada then turns her gun on him, having blown her cover, but is unable to pull the trigger due to latent feelings she has for him. Annette instead shoots her in the shoulder, and she falls off the ledge as the facility begins to collapse. Annette then appears to die from internal hemorrhaging.
As Leon travels down to an emergency train at the bottom of the facility, and finds out in a computer room that Claire has already reached the train at the bottom. On his way he is stalked by T-00, still intent on killing him. As he is grabbed by the head, debris sends him falling down another level, but he finds an elevator to take him further down. T-00 returns, having been injured by the debris, and begins mutating into a more powerful form without its Power Limiter. Leon's weapons have little effect, and he can only kill the Tyrant with a rocket launcher thrown down to him by a mysterious stranger. At the bottom, Leon rushes onto the train, which he finds is already being crewed by Claire and a young girl named Sherry. The train is rocked by something, and Leon goes to the rear car to investigate. William returns, now in its G5 form. A giant blob with teeth, it forces its way into the car slowly, ripping it to pieces. After the train roof is thrown off, Claire and Sherry come to investigate, and Claire tries to unbuckle the carriage so it can be consumed by the oncoming explosion from NEST. Leon stabs William in the eye and is helped onto the middle carriage.
Arriving at Umbrella's train stop at the other end, the three get off and begin walking through the long, flat landscape of Arklay County on the other side of the mountains. Sherry is confused about how Claire and Leon know each other, and presumes them to be partners. The three spot a tanker truck coming towards them, and Claire and Sherry hide while Leon deals with it. The driver flips Leon off for standing in the middle of the road and continues driving, having no idea what happened to Raccoon City. Claire and Sherry get back from hiding, and the three walk off into the distance, with Sherry asking if she is going to be adopted and if they'll buy her pets.