In Night at the Museum: Battle of the Smithsonian, players assume the role of Ben Stiller's character, Larry Daley, former night guard of New York's Museum of Natural History. Larry must rescue Jedediah and his other friends from Akhmenrah's brother who captured them while stealing a powerful Magic Tablet that can raise the Army of Horus to take over the world. Daley is well aware of the havoc that history's most notorious figures can wreak when the sun goes down and the museum exhibits come alive. He races to DC to help his friends in the Smithsonian.

Using Larry's trusty key chain and flashlight, players must solve puzzles and adventure through 14 levels including the Federal Archives, the National Air and Space Museum and the Lincoln Memorial. Gamers can travel in style on the back of a T-Rex skeleton, within the Lunar Lander and in the cockpit of Amelia Earhart's Pitcairn Autogyro. Along the way, players must find pieces of the Magic Tablet and collect other items to unlock mini-games, new areas and bonus content from the Smithsonian including trivia questions, in-game audio tours and factoids.