The Microsoft co-founder and his wife, who launched the world's largest charitable foundation, said they would continue to work together at the Bill & Melinda Gates Foundation.
In identical tweets on Monday, they said they had made the decision to end their marriage of 27 years.
"We have raised three incredible children and built a foundation that works all over the world to enable all people to lead healthy, productive lives," they said in a statement.
"We ask for space and privacy for our family as we begin to navigate this new life."
In her 2019 memoir, The Moment Of Lift, Melinda Gates wrote about her childhood, life and private struggles as the wife of a public icon and stay-at-home mother with three kids.
She won Gates' heart after meeting at a work dinner, sharing a mutual love of puzzles and beating him at a maths game.
The couple's sprawling Seattle-based foundation is easily the most influential private foundation in the world, with an endowment worth nearly $US50 billion ($A64 billion). It has focused on global health and development and US education issues since incorporating in 2000.
The couple were married in 1994 in Hawaii. They met after she began working at Microsoft as a product manager in 1987.
Last year, Bill Gates, formerly the world's richest person, said he was stepping down from Microsoft's board to focus on philanthropy.
Gates was Microsoft's CEO until 2000 and since then has gradually scaled back his involvement in the company he started with Paul Allen in 1975.
He transitioned out of a day-to-day role in Microsoft in 2008 and served as chairman of the board until 2014.
The Gateses will be the second high-profile Seattle-area billionaire couple to end their marriage in recent years.
Amazon CEO Jeff Bezos and MacKenzie Bezos finalised their divorce in 2019. MacKenzie Scott has since remarried and now focuses on her own philanthropy. She received a 4 per cent stake in Amazon, worth more than $US36 billion ($A46 billion).