With the donation, officials say the museum's capital campaign has raised $111 million toward its $150 million goal. The new museum is set to open in 2010.
Spielberg helped establish the Righteous Persons Foundation in 1994 after directing his Oscar-winning Holocaust film "Schindler's List."
The museum was established in 1976 and is dedicated to telling the story of the American Jewish experience. It is constructing the new building in hopes of raising its profile and increasing the number of visitors.