It's been several years since Larry Daley (BEN STILLER) worked as a night guard at New York City's Museum of Natural History. Despite now being a successful infomercial entrepreneur and product inventor, Larry misses his former friends at the museum, but he can only visit them at night.
That's because thanks to an ancient Egyptian tablet with magical powers, various figures there -- Attila The Hun (PATRICK GALLAGHER) and his crew, Lewis and Clark's Indian tracker Sacajawea (MIZUO PECK), miniature diorama figures cowboy Jedidiah (OWEN WILSON) and Roman emperor Octavius (STEVE COOGAN), and former President Teddy Roosevelt (ROBIN WILLIAMS), not to mention several Neanderthals, a T-Rex skeleton and a capuchin monkey known as Dexter -- come to life at night after the museum is closed.
Things are going to change, however, due to the museum undergoing renovations where most of the figures will be sent to the Federal Archives located underground beneath the Smithsonian museums in Washington, D.C. And with the magical tablet staying put, that means those figures will never come to life again. Accordingly, when Larry gets a call for help from Jedidiah, he decides to travel to D.C. to set things straight.
After stealing a security pass, he enters the underground complex only to find Egyptian pharaoh Kahmunrah (HANK AZARIA) -- the older jealous brother to his 3,000-year-old counterpart in the New York museum -- has likewise come to life thanks to Dexter the monkey taking the magical tablet with him. Kahmunrah wants to resurrect his army of the undead, but Larry and newly animated fly girl Amelia Earhart (AMY ADAMS) manage to elude and leave his guards inside a painting.
Accordingly, Kahmunrah enlists the likes of Ivan the Terrible (CHRISTOPHER GUEST), Napoleon Bonaparte (ALAIN CHABAT) and Al Capone (JON BERNTHAL) as his new minions whose task is to catch Larry and force him to decipher the Egyptian tablet's secret code. That's all while Larry and Amelia get help from General George Armstrong Custer (BILL HADER) and gigantic stone President Lincoln (HANK AZARIA) from his monument to try to thwart the new museum villain.