Aspiring New York City inventor Larry Daley bounces between jobs and apartments. Although sympathetic, his ex-wife Erica believes he is a bad example to their ten-year-old Nick, who considers quitting ice hockey and plans to work in the finance sector like Erica's fiancé Don. Larry fears Nick admires Don over him, especially as Don gets invited to his school's Career Day. Larry interviews at the Museum of Natural History with the almost retired night security guard Cecil Fredericks. As the museum is hemorraging money, Cecil and his colleagues Reginald and Gus are being replaced with one guard, so Larry gets hired despite his resume. Cecil gives him a manual, warning him he "not let anything in... or out." On his first night, Larry falls asleep. Waking, he discovers the T-rex skeleton is missing. After finding the T-rex alive and being chased by him, he calls Cecil, who suggests using the manual. Larry tames him by throwing a bone, then names him "Rexy". After sunset, all exhibits come alive, including: Dexter, a mischievous stuffed capuchin monkey who destroys the instructions and takes Larry's keys; rival miniature civilizations depicting the American Frontier, Ancient Rome, and Ancient Maya; a chewing gum-loving Easter Island Moai who calls Larry "Dum-Dum"; American Civil War soldier mannequins; and wax models of Attila the Hun and his men, pyromaniacal Neanderthals, and Sacagawea. A horse-mounted Theodore Roosevelt rescues Larry from feuding miniature leaders Jedediah and Octavius. He explains that ever since the 1952 arrival of Egyptian Pharaoh Ahkmenrah’s Golden Tablet, the exhibits come alive each night. All outside at sunrise disintegrate. As they restore order, Larry sees Theodore loves Sacagawea but is too shy to tell her. The retirees check on Larry, who almost quits, but Nick and Don's congratulatory visit convinces him to stay. Cecil suggests he read up on history. Larry controls the exhibits better, but must extinguish a fire the Neanderthals start. Meanwhile, Dexter steals his keys again, unlocking a window, so a Neanderthal escapes onto the street, seeing a group of indigents with a fire pit, and disintegrates in the sun. Nick witnesses museum director Dr. McPhee firing Larry over the damaged Neanderthal exhibit. Eventually Larry is given another chance to prove himself. Rebecca Hutman, a museum guide and historian writing her dissertation on Sacagawea, believes Larry is mocking her when he reveals the museum's nighttime secret. Larry wants Nick to see the T-Rex come alive, but nothing happens. The tablet has been deactivated by the retirees who want to steal it with other valuables to fund their retirement and frame Larry. They want the tablet for its vitality restoring powers. Having regained and reactivated the tablet, Larry and Nick are chased through the museum, until Cecil snatches it, locking them in the Egyptian room. Larry releases Ahkmenrah's mummy from his sarcophagus. The pharaoh reveals himself as a fully living human, and helps them escape. Larry convinces the squabbling exhibits to help catch the guards and recover the tablet. Gus and Reginald are captured, while Cecil escapes via Pony Express stagecoach. Theodore pushes Sacagawea out of Cecil's path and is sliced in half but survives, being "made of wax." Larry pursues Cecil into Central Park, regaining the tablet. Theodore bonds with Sacagawea as she repairs him. Rebecca sees the exhibits returning to the museum and realizes Larry was being truthful; he later introduces her to Sacagawea. Theodore and Nick are proud of Larry's work. Larry laments he could not save everyone, believing that Jedediah and Octavius were both killed, so is surprised upon their return. The next day, McPhee attempts to fire Larry after news reports about the night's strange events, such as The Neanderthals leaving cave paintings in the museum's subway station and Rexy's dinosaur tracks in Central Park, but the publicity boosts museum attendance. Larry is rehired, goes to Nick's next school career day, and celebrates that night with a party, with Nick, Rebecca and the exhibits. In a post-credits scene, it is revealed that the former guards, rather than being turned in to the police by Larry, must work as museum janitors as punishment for their actions, so they clean up after the party.
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