The 59-year-old will have the Washington DC facility's 350-seat theatre named after her as a special thanks, the Hollywood Reporter said.
"I am so proud of African American history and its contributions to our nation as a whole. I am deeply appreciative of those who paved the path for me and all who follow in their footsteps," Winfrey said.
"By investing in this museum, I want to help ensure that we both honour and preserve our culture and history, so that the stories of who we are will live on for generations to come," she said.
Lonnie G Bunch III, the museum's founding director, said, "We are inspired and profoundly grateful for Winfrey's generosity at this important time. Her gifts will forever be associated with harnessing the power of art and creative expression to build bridges between cultures and enrich people's lives."
The National Museum of African American History and Culture is currently under construction and is set to open in late 2015.