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"After careful consideration and a lot of hard work on our relationship, we have decided to end our marriage," the Washington state couple said in a joint statement on Twitter.
The Gates, who have three children, announced that they will continue to "work together on their foundation," the Bill and Melinda Gates Foundation, which fights poverty and disease, but no longer believe they "can grow together as a couple."
Bill Gates, 65, and his wife, 56, asked the public to give their families the "space and privacy" they need to "begin to adjust to this new life."
They said nothing about the reasons behind their separation.
Bill Gates, according to the latest Forbes business magazine ranking, is the fourth richest man in the world, with a fortune estimated at $124 billion. That puts him behind two other Americans, Jeff Bezos and Elon Musk, Frenchman Bernard Arnault, and ahead of Facebook founder Mark Zuckerberg.
Although he made his fortune through Microsoft, he stepped down as chairman in 2014 and now devotes himself mainly to philanthropy.
The foundation he created with his wife is considered the largest private foundation in the world. It has spent some $53 billion in 20 years and employs nearly 1,600 people, according to its official website.