The 65-year-old Microsoft founder announced the split on Twitter.
"After a great deal of thought and a lot of work, we have made the decision to end our marriage. Over the last 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," the couple said in a joint statement.
"We continue to share a belief in that mission and will continue to work together at the foundation, but we no longer believe we can grow together as a couple in this next phase of our lives.
Bill and Melinda got married on New Year's Day in 1994, in a private ceremony in Hawaii.
In 2000, they launched the Bill & Melinda Gates Foundation, with ambitious goals that included the reduction of extreme poverty around the world.
This past year, it was heavily involved in the world's response to Covid-19, committing $1.75b to help develop and distribute vaccines.
The couple met in 1987, when Mrs Gates - then known as Melinda French - was a product manager at Microsoft.
In a Netflix documentary series a couple of years ago, Melinda shared an amusing anecdote from about year into their relationship, when she walked in on Bill as he was drawing up a list of the "pros and cons" of getting married.
Melinda Gates and Bill Gates have announced their split online. Photo / Getty Images
"He had to make a decision," she said, bursting into laughter.
At first, the pair revealed, their relationship was a casual one.
"She had other boyfriends, and I had Microsoft," said Bill.
"We were like, 'Hey we are not really serious about each other, are we? We are not going to demand each other's time.'"
"I was new to Microsoft, there were a lot of men there, and you are still looking around," Melinda added.
