Supreme Court Chief Justice John Roberts and members of Congress were also there to see the movie, a sequel to 2006's "Night at the Museum."
Stiller, who starred in both movies, said he was very happy with the way the sequel turned out.
"The Smithsonian was the springboard for doing the whole thing again," Stiller said.
It's the first major Hollywood premiere at the Smithsonian.
In the film, Stiller's character, security guard Larry Daley, comes to Washington to find his museum friends from the first movie, who had been shipped from New York to a mythical vault under the National Mall.
"Creatively, I think it turned out better than the first movie," Director Shawn Levy said.
After the first film, attendance shot up at New York's American Museum of Natural History, and Smithsonian curators hope the same thing will happen there.
"This is really bringing the Smithsonian to life for a new audience," said U.S. Rep. Doris Matsui, D-Calif., who is on the Smithsonian's board. "A lot of people haven't been here yet."
The Smithsonian Institution and 20th Century Fox have inked numerous promotional deals and have plans for a slew of movie-themed products. On Wednesday, game maker Majesco Entertainment Co. released the first video game to explore the halls of the Smithsonian after dark, following the movie's plotline.
The movie opens nationwide May 22.
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