In Night in the Woods, the player controls a character named May. She is an ordinary teenage girl who is sick and has a dissociative disorder that does not let her recognize other people or animals except for rectangular shapes. She has no memory of her childhood. She was born in the year 2000 and has no memory of her parents.

May is in her hometown of Possum Springs, which is a small city in the middle of the woods. In the game, she is the only human being who can see the animals of her childhood, yet she does not know them. She also has friends, named Nick and Dave, who are also in the town. May goes to the town to talk to her friends and get back to her old life, yet this is no more. The town's residents start talking about something in the nearby forest.

May goes out to find out what's going on. She gets her friends together and they go out to find out what's going on. The game focuses on the storyline of the game, rather than the gameplay experience.