Two years after the events of the first film, Larry Daley, former night guard at the American Museum of Natural History, now runs his own direct response television company that sells inventions based on his experiences. He travels to the museum, discovering most of the exhibits will be moved to the Federal Archives at the Smithsonian Institution and replaced with holographic information providers. The Tablet of Ahkmenrah is to remain in the museum, leaving most of the exhibits without the ability to come to life at night. After the exhibits are moved, Larry receives a phone call from Jedediah, who informs him that Dexter the monkey stole the tablet and took it to the Smithsonian, bringing every exhibit in it to life. Larry travels to Washington, DC, navigating his way to the archives with help from his son Nick while posing as a night guard.

Larry Daley and his heroic friends embark on the greatest adventure of their lives, as they travel to London to save the magic that, after dark, allows the museum's exhibits to come to life.