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- Isaac Clark: The main character of the game. Isaac is a systems engineer traveling to repair the USG Ishimura, in company with three security personnel and a computer specialist. Unfortunately for them, they are thrust into the middle of the nightmare that the USG Ishimura has become. Clarke is also trying to find his Ex-girlfriend, Nicole, a medical specialist assigned to the ship.
- Kendra Daniels: Kendra is a technology expert and part of the team sent aboard the Ishimura once the ship goes silent. She helps the player through the Ishimura in many ways, giving directions and assisting through areas of the ship that would otherwise be inaccessible.
- Zach Hammond: The senior security officer who travels with both Isaac and Kendra to the Ishimura. Hammond's initial desire to complete the original mission and not immediately abort puts him at odds with Kendra, who finds this suspicious. Throughout the game he works to fulfill their original mission, but as time goes on, he becomes more disillusioned and focuses instead on getting himself and the rest of the surviving team off the ship.
- Dr. Challus Mercer: A doctor aboard the USG Ishimura and a devout Unitologist. In the wake of the calamity that has gripped the Ishimura, Mercer has become a religious fanatic, and attempted to convince the crew of the Ishimura to commit mass suicide. Mercer often works to bar the progress of Isaac and even attempts to kill him outright.
- Dr. Terrence Kyne: The Chief Science Officer of the Ishimura who appears later in the game and asks for help from the surviving members of the Kellion team. He seems to be a bit eccentric, perhaps deranged, but offers to aid Isaac in stopping the nightmare that has gripped the Aegis System and the Ishimura.
- Nicole Brennan: Isaac's ex-girlfriend and a medical specialist assigned to the Ishimura. She sent him a video asking for help and is one of the reasons he came to the Ishimura.
- Jacob Temple: Jacob is an engineer on the USG Ishimura who is, like Isaac, trying to find his own girlfriend, Dr. Elizabeth Cross. The player often finds audio logs left behind by him as he progresses throughout the game.
- Elizabeth Cross: Originally assigned as a herbologist aboard the USG Ishimura, she is looking for her boyfriend, Jacob Temple. Throughout the Ishimura you can find various audio logs, particularly in the herbology deck.
- Corporal Chen: Pilot of the shuttle which crashes onto the USG Ishimura, and one of the security troopers who comes aboard under Hammond.
- Corporal Johnston: The co-pilot of the repair shuttle. Johnston is also under the command of Hammond.
- Captain Benjamin Matthius: The captain of the Ishimura.