In the vibrant heart of New York City, we meet Larry Daley (Ben Stiller), a divorced and aspiring inventor grappling with his identity while drifting through unfulfilling jobs and unstable living conditions. His ex-wife, Erica, despite her empathy, sees him as a troubling influence on their ten-year-old son, Nick—a boy already taking to finance, inspired by his mother's fiancé, Don. Larry is increasingly worried about his diminishing role in Nick's life, especially heightened when the boy invites Don to Career Day at school, a move that threatens to further cement Don’s place as his role model.
As Larry wades through these turbulent waters, he snags a position as a night security guard at the Museum of Natural History, taking over from the outgoing guards—Cecil Fredericks, Gus, and Reginald. Larry's first night on the job comes with a thorough manual from Cecil, which emphasizes the importance of watching over both the exhibits and the museum's surroundings—a responsibility that soon proves to be more complicated than he imagined.
On his very first night, Larry is thrust into a world of unexpected confrontations as the museum’s exhibits spring to life after dark. From the playful antics of a Tyrannosaurus skeleton named Rexy to a capuchin monkey Dexter, known for his penchant for mischief—including stealing Larry's keys—the chaos escalates quickly. Larry discovers intricate miniature civilizations within the museum, showcasing cultures from Ancient Rome, the Old West, and Ancient Maya. Among the lively exhibits, a Moai from Easter Island enjoys chewing gum while wax figures—ranging from Attila the Hun to Civil War soldiers and fire-loving Neanderthals—add to the whimsical madness of the night.
Just as things spiral out of control, Larry receives a savior in the form of Teddy Roosevelt (Ricky Gervais), who reveals that the night-time antics are the result of an ancient Egyptian artifact, the Golden Tablet of Pharaoh Ahkmenrah, which has granted life to the exhibits since it was brought to the museum in 1952. Teddy warns Larry that if these exhibits are not kept within the museum by sunrise, they will disintegrate, adding weight to Larry's newfound responsibilities.
As Larry grapples with his duties, he uncovers Teddy's secret crush on Sacagawea, a historical figure trapped in glass, likewise becoming entangled in Larry's evolving predicament. A surprise visit from Nick and Don temporarily lifts Larry’s spirits, and with encouragement from Cecil, he delves into the history around him—a decision that leads to more hilarity and misadventures. When the Neanderthals inadvertently start a fire, it falls on Larry to extinguish it, while Dexter's theft of his keys leads to a Neanderthal's escape.
As morning breaks, Larry feels the weight of defeat, culminating in the disintegration of the runaway Neanderthal and his dismissal from the museum by the demanding Dr. McPhee. Enter Rebecca Hutman, a sharp-minded historian with a passion for Sacagawea, who becomes embroiled in Larry's fantastical troubles when he reveals the museum’s nighttime secrets. Her skepticism gradually gives way to a deeper understanding as Larry takes Nick for a special tour, only to find those they thought trustworthy—Cecil, Gus, and Reginald—plotting to exploit the tablet's powers for themselves.
When Nick inadvertently activates the tablet, it sends the museum into disarray. In a frantic chase, Larry and his allies attempt to retrieve the tablet from the treacherous trio, but they find themselves locked in the Egyptian room. It is then that Larry calls upon Ahkmenrah's ([actor name]) mummified form for assistance, facilitating their escape amid the chaos of engaged artifacts battling for essence and dominion.
Larry seizes the moment to unite the exhibits against their felonious foes, transforming the museum into a battleground, drawing upon Ahkmenrah's wisdom to reclaim the tablet and restore harmony in their beloved institution. As the dust settles, Cecil, Gus, and Reginald find themselves facing the consequences of their actions. In a heroic moment, Teddy sacrifices himself for Sacagawea, but against the odds, he survives—wounded but with newfound resilience.
Larry’s pursuit of justice leads him into a thrilling chase through Central Park, where he finally triumphs over Cecil and reclaims the sacred tablet. As balance is restored, Rebecca gains insight into the strange happenings, guided by Larry as she meets Sacagawea. However, the following day brings unexpected challenges when Dr. McPhee attempts to fire Larry for the prior night's bizarre incidents—ironically, these events have surged attendance at the museum, resulting in Larry’s reinstatement amid cheers.
As night falls once more, a festive gathering unfolds among Larry, Nick, Rebecca, and the once mischievous exhibits. With a poignant touch, Cecil, Gus, and Reginald, now reduced to janitors, scrub away the remnants of their folly—a sharp reminder of the lessons learned amidst the adventures that turned their lives upside down.
