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Robert NevilleThe protagonist, and as far as he knows, the only survivor of the plague. Once a factory worker, Neville now spends his days hunting vampires or repairing his house, while hiding in his home at night. Due to months and then years of isolation, Neville grows increasingly depressed and unstable.
- Action Dad: He was a father, but his daughter, Kathy, died from the plague. He had to have her body burned in order to prevent reanimation.
- Action Survivor: Neville is tough, but his still an average person. The reason he can so easily kill or capture vampires is because they're in a coma, or sleeping very heavily, during the day. He is captured relatively easily by members of the new society.
- The Alcoholic: At first, in order to cope with his isolation. He eventually stops for reasons he cannot reasonably explain.
- Badass Beard: Ends up growing one, due to the lack of need to shave.
- Badass Bookworm: Starting from Part II, he spends a lot of his time studying the bacteria, trying to find a cure, or experimenting on vampires (living or dead). He is not an expert in any of this, however.
- Driven to Suicide: Neville takes many risky actions throughout the story, such as fighting a horde of vampires or drinking heavily. He always stops before it is too late, however. He kills himself using pills given to him by Ruth, because he fears a violent, painful execution.
- Good Is Not Nice: Neville lost most of his social skills due spending years by himself, but he is still a good person at heart and is willing to accept being punished by the new society for his crimes.
- Happily Married: To Virginia.
- Heartbroken Badass: The death of Virgina and Kathy hit Neville hard, and he thinks about them frequently throughout the story.
- Hoist by His Own Petard: His indiscriminate killing of living and dead infected turn the former against him, leading to his capture and death.
- Last of His Kind: Neville is the last uninfected human known, both to himself and the new community of infected humans.
- The Immune: To the vampire bacteria.
- Sanity Slippage: Throughout the story, though he seems to grow more stable at the end.
- Vampire Hunter: When not working on his house, Neville spends his day looking for supplies and killing sleeping vampires.
- "Come out, Neville!"
- Alas, Poor Villain: Neville has this reaction to his being shot at and eventually killed by the vampire hunters.
- Cannot Cross Running Water: Subverted. Neville tries to see if the legend that vampires cannot cross running water is true. Cortman being Cortman, upon noticing the hose, jumps across it again and again in order to mock Neville.
- Celebrity Resemblance: Neville mentions he looks like Oliver Hardy several times.
- Climbing Climax: He tries to escape from the new society's soldiers by climbing up and hiding in a chimney. He fails, eventually being knocked off the roof due to machine gun fire. He is then stabbed with a lance, which presumably kills him.
- Evil Former Friend: Cortman was a neighbor and friend to Neville. After Cortman returned from the dead, he began hunting Neville.
- Fat Bastard: He is described as being "portly."
- Say My Name: When he can see Neville, he will just shout "Neville!" It appears to be one of the only three words he can say.
- Too Dumb to Live: He tries to hide in his chimney when soldiers begin killing vampires outside Neville's house, even after they see him climbing up the house, have a spotlight on him, and a firing upon him. Despite all his cleverness, he is still just one of the dead vampires.
- Laughably Evil: Ben is often whimsical in his behavior, often teasing or laughing at Neville.
- Worthy Opponent: Neville seems to think of him this way, at least. Neville remarks he doesn't deserve to be butchered by the new society's vampire hunters.
The DogA dog that Neville notices around his house. Due to years of isolation, Neville tries to befriend it, but it is terrified of him.
- Canine Companion: Subverted. Neville tries to take the dog as a per, but it is too scared of him and refuses to let him touch it. It eventually starts to trust Neville a bit more, but ends up getting infected before completely warming up to him.
- Post-Apocalyptic Dog
- Zombie Infectee: It disappears for a few days, and when it returns, it is implied to have been bitten. Neville tries to save it, but it dies after a week.
RuthThe only uninfected survivor Neville comes across. She claims to have lived with her husband until he was killed by vampires a few weeks ago. Initially distrustful of Neville, she eventually grows closer to him.
- Bad Liar: Ruth isn't a particularly convincing liar, and she appears to make up her story as she goes. Neville continuously tells himself that he is just being paranoid whenever he finds a hole in her story.
- Becoming the Mask: Ruth ends up caring for Neville, even though he killed her husband, after learning that he is not really a monster or even a particularly cruel person. She leaves him a note, begging him to escape before he is captured, and gives him pills that would kill him in order to spare him a violent execution.
- Daywalking Vampire: Ruth is infected just like everyone else, but it capable of walking in the sun for short periods of time due to medication.
- Did Not Die That Way: Not Ruth herself, but her husband. She claims he was killed by vampires, but he was actually killed by Neville.
- Friendly Neighborhood Vampire: Ruth is shown to be a kind person, despite being infected. Justified in that most living infected are otherwise normal people, particularly after they begin taking medication.
- Obfuscating Stupidity: She asks Neville a lot of questions about the vampires that he thinks would be obvious, and that she would have to know to survive. It turns out she was most likely just testing his knowledge on the bacteria.
- The Mole: For the new society.
- There Is Another: The only uninfected human Neville encounters in the story. Subverted, as it turns out she is infected, just like everyone else in the world. Aside from Neville, that is.
- Vegetarian Vampire: Like the rest of her people, she feeds the vampire bacteria with a pill that contains blood.
- Vampire Infectee: Like every other human in the world, except for Neville. She and the rest of her people take medication to prevent its growth, however.
- Big "NO!"! When Sam is injured protecting him
- Dying Moment of Awesome: But only in the theatrical ending, where he runs head-first into the zombie horde and explodes himself on them so Anna could escape with the cure. In the alternate ending, he lives to see that the zombies were really only trying to rescue one of their kind.
- Good Is Not Nice: At least not always.
- Heartbroken Badass: His wife and kid died in a helicopter accident as they were trying to escape New York City.
- The Kindnapper: Uses one of the zombies as a test subject for his research, not knowing that the zombies are sapient enough to treat this as kidnapping.
- Mad Scientist: Notable for not being evil.
- Science Hero: Deconstructed. His main goal is to cure the disease and reverse the zombie apocalypse, all the while unaware of how sentient the zombies actually are.
- Unstoppable Rage: When Sam dies.
- Wrong Genre Savvy: Is trying to make a cure for the zombie apocalypse despite zombies having become the norm, and being more sentient than Neville gives them credit for...
- This Is My Human: Is revealed to see Neville as this after getting injured by the zombie dogs.
- Tragic Keepsake: She's the only thing that Neville has left of his family.
- Action Girl: Implied to be this offscreen
- Authority Equals Asskicking: The leader of the pack amongst the Darksiders, and is not above getting into the thick of it, or smashing his underlings out of his way.
- Big Bad: The main antagonist of the film.
- It Can Think: He, like the rest of his kind, are a lot smarter than Neville gives them credit for.
- Love Makes You Crazy: As shown in the original ending, his fury at Neville and determination to hunt him down are motivated by a desire to reclaim his mate.
- Roaring Rampageof Revenge: When Neville kidnaps his mate, the Alpha goes on one to get her back.