The Amazon boss, 54, said he and novelist MacKenzie, 48, mum of his four kids, have decided to split for good after a trial separation.
It raises the prospect of the world's richest divorce as Bezos's wealth has soared to an estimated $160bn (£125bn) thanks to his stake in retail giant Amazon.
Bezos posted a joint statement on his Twitter account "to make people aware of a development in our lives".
It said: “As our family and close friends know, after a long period of loving exploration and trial separation, we have decided to divorce and continue our shared lives as friends.
"We feel incredibly lucky to have found each other and deeply grateful for every one of the years we have been married to each other."
The statement went on: "If we had known we would separate after 25 years, we would do it all again.
"Although the labels might be different, we remain a family and we remain cherished friends.”
Forbes magazine's "real-time rich list" currently puts his personal fortune at $136.4bn (£107bn), after he became the first man ever to have a wealth of more than $150bn.
Reuters estimates his wealth at more than $160bn thanks to his 16 per cent holding in Amazon, the online retailer he founded in his garage in 1994.
Amazon.com once again became Wall Street's most valuable company this week, surpassing Microsoft.
He also owns Blue Origin, the space company Bezos claims will blast tourists into orbit next year.
Jeff and MacKenzie reportedly met when they both worked at a New York hedge fund and wed six months later, in 1993.
Jeff interviewed her for her job there. He later joked to Vogue: "I think my wife is resourceful, smart, brainy, and hot, but I had the good fortune of having seen her résumé before I met her, so I knew exactly what her SATs were."
He quit finance months after their wedding and moved to Seattle where he started the company that now dominates shopping across the world.
MacKenzie was one of Amazon's first employees and has been by Jeff's side as he built the firm into a global giant that now incorporates streaming and cloud data services.
She told CBS in 2013: "To me, watching your spouse, somebody that you love, have an adventure — what is better than that?"
Last year Bezos was described as the richest person in history after his fortune nearly doubled along with the value of Amazon, the second ever trillion-dollar company after Apple.
He and his wife own more land than almost anyone else in America, reports Business Insider.
Last year the couple set up a charitable fund called the Day One Fund. Their statement suggests they will continue to work on projects together.
Bezos also owns the Washington Post newspaper, prompting vicious attacks from Donald Trump who slammed Amazon for not paying enough tax.
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