The 59-year-old star will have the facility's 350-seat theatre named after her as a special thanks, reports femalefirst.co.uk.
“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,” said Winfrey.
"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 added.
Winfrey, who has been a member of the museum's advisory council since 2004, previously donated one million pounds to the museum in 2007.
Lonnie G. Bunch III, the museum's founding director, said: “We are inspired and profoundly grateful for Ms. 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,” he added.
The National Museum of African American History and Culture is currently under construction and is set to open in late 2015.