The media mogul is donating $12 million to the National Museum of African American History and Culture.
The museum says she has already given a $1 million donation, making her $13-million contribution the largest to date.
Construction on the $500 million museum began in early 2012. When it's finished in 2015, the museum will be the 19th Smithsonian museum. The U.S. government is providing half of the funding. To date, about $140 million has been raised in private funds.
The museum's theater will be named after Winfrey.
"I am deeply appreciative of those who paved the path for me and all who follow in their footsteps. By investing in this museum, I want to help ensure that we both honor and preserve our culture and history, so that the stories of who we are will live on for generations to come," Winfrey said in a statement released by the museum.
The museum is also in talks to acquire memorabilia from her career.
The Associated Press contributed to this report.