Larry Daley. A struggling entrepreneur who can't seem to stay at one job or business for long. His wife, exhausted by the situation, divorced him, and he lives with his son, Nick. As time passes, he decides he doesn't want to show this side of himself to his son anymore and searches for work, eventually getting hired at the financially struggling New York Museum of Natural History. Although he is a security guard, he secretly steals uniforms and key cards from other museum guards to climb over walls or sneak into other museums. The reason, of course, is the Tablet of Ahkmenrah.

Teddy Roosevelt. The 26th President of the United States. However, since he is currently a wax figure, his body parts can be reattached using fire. He is an excellent leader who gives advice to the wandering Larry and commands the rowdy exhibits all at once. Yet, he has a timid side, unable to confess his feelings to Sacagawea, whom he has a crush on. He appears not as the President, but as a U.S. Cavalry officer. He is on display near the entrance, mounted on his horse.

Sacagawea. She served as a guide and interpreter for explorers Lewis and Clark on their journey to the Pacific. She is also a wax figure rather than a real person. Like the others, she can reattach fallen body parts using fire. Communication is difficult because she is behind glass that blocks sound. Even when she speaks, it's hard to hear her. She is very helpful to Larry.

Attila. King of the Huns, he treats Larry roughly, even threatening to tear him limb from limb. He seems to like magic. Larry tries to show him a magic trick, but when the trick is exposed, Attila becomes enraged and nearly has Larry executed by dismemberment. He wears Hunnic armor and has a thick mustache. He communicates in an unknown language.

Ahkmenrah. The fourth Pharaoh of Egypt, he ascended to the throne at a young age. He is the owner of the tablet that brings everything to life. Before Larry opened his sarcophagus, he had been trapped inside for 54 years, doing nothing but struggling to get out. Although he is from Egypt, he was once exhibited in Britain, so he is fluent in English. He not only speaks English but also acts as an interpreter between Attila and Larry. He plays a leadership role, stopping the exhibits from fighting. He is very friendly and is on display in the Egyptian wing.

Dexter. A brown capuchin monkey with incredibly fast hands and feet. He dislikes Larry, doing things like snatching Larry's keys and manual in an instant or even peeing on him. However, he behaves well for everyone except Larry. He has a cute smile.

Rexy. A Tyrannosaurus Rex skeleton and the first exhibit you see upon entering the lobby. He is quite fierce and nearly killed Larry. However, when one of his ribs falls off, he plays with it or begs Larry to play fetch. He currently follows Larry well.

Tigers, ostriches, lions, whales, and more. There are various species divided into land and sea creatures. Most of them are not friendly.

The former security guards. All three are elderly men: Gus, Cecil, and Reginald. All three attempt to lock Larry up and steal the museum's artifacts.

Mainly representative figures: Jedediah from the West and Octavius from Rome. They each wear clothes representing their respective eras. They constantly insult and fight each other. It's said they fight every single night. They aren't particularly friendly toward Larry. Other exhibits include the Moai statue, Columbus, Civil War mannequins, and Neanderthals. The Civil War soldiers are mannequins and thus have no facial features; they engage in hand-to-hand combat. The Moai loves gum and repeats words ending in 'M' or 'N' twice. He has a loud voice that can stop a commotion instantly. Columbus speaks in Italian and waits for Larry to understand his language; if Larry doesn't, he gets sulky. He warns Larry of various dangers. The Neanderthals revere fire and try to start fires everywhere in the museum. They eat fire extinguisher powder and make 'ooga-booga' sounds because they don't know the language.