Wendi Deng gained popularity for springing to her husband's aid when a protester threw a custard pie at him as he appeared before MPs to answer questions about phone hacking in 2011.
At 44, Ms Deng is 38 years younger than Mr Murdoch, who is 82.
Rupert Murdoch's divorce filing said the "relationship between husband and wife has broken down irretrievably," according to a New York Times report.
It added that a spokeswoman for News Corporation, his media company, confirmed that it was Mr Murdoch who had made the filing.
The spokeswoman added that the divorce would have no impact on the company.
Rupert Murdoch's first marriage to flight attendant Patricia Booker took place in 1956 and lasted 11 years. They had one daughter, Prudence, in 1958.
In 1967, he married the Scottish-born journalist Anna Torv after the pair met at his Sydney-based newspaper The Daily Telegraph.
They had three children together - Elisabeth, Lachlan and James - over the course of more than 30 years of marriage.
Within a month of their divorce in 1999, Murdoch married Chinese-born TV executive Wendi Deng with whom he had two more daughters - Grace and Chloe.
Wendi Deng is perhaps best known in Britain for defending husband Rupert during a physical attack at a parliamentary hearing in 2011.
The Chinese-born businesswoman was seen landing a slap on the face of activist Johnnie Marbles, who had attempted to throw a foam pie at Mr Murdoch.
News Corporation chief executive Rupert Murdoch has filed for divorce from his wife Wendi, the company confirmed.
Murdoch, 82, married Wendi Deng, his third wife, in 1999. The reasons for the filing were not immediately clear.
Media tycoon Rupert Murdoch claimed Britain is "far more successful" because of Baroness Thatcher's "brave leadership".
Mr Murdoch praised the former Prime Minister's role in facing down the trade unions in the 1980s and said the Government made it possible for News International to survive a year of industrial action fighting against a move of operations to Wapping, East London.
The tycoon wrote in The Times (£): "Mrs Thatcher understood that risk was a vital ingredient in a free enterprise society. She understood that such a society had to be led by a government with backbone.
"After the Second World War, in which the country lost a second generation of its finest men, Britain had created a dependency state. It killed off aspiration.
"In 1979 Margaret Thatcher set about its rehabilitation. She put the economy on a sound footing, she ended a culture of crippling strikes, she encouraged entrepreneurs to come here and set up their businesses.
"Thanks to her I have experienced in Britain many of my defining moments as a businessman, a Britain that is far more successful as a result of her brave leadership."
Mr Murdoch tweeted this evening about how Nigel Farage, whose party finished a shock second in the Eastleigh by-election, was "reflecting opinion":
Few days in UK, Italy. Politics both places very fluid, economies going nowhere. New leaders emerging on distant horizon.
Boris Johnson, Michael Gove, few excellent, frustrated ministers. Farage reflecting opinion. Florence mayor Renzi brilliant young Italian.
News Corp boss Rupert Murdoch has hinted that he believes Page 3 of The Sun may be becoming outdated.
Responding to an anonymous Twitter user who said it is "so last century", Mr Murdoch said he was "considering" whether he agreed.
@kazipooh page three so last century! You maybe right, don't know but considering. Perhaps halfway house with glamorous fashionistas.
Page 3 of the tabloid has long been reserved for pictures of topless or scantily-clad woman.
News Corporation chairman Rupert Murdoch has suggested hackers are "still" trying to gain access to his companies' servers.
Chinese still hacking us, or were over weekend.
News Corp's Wall Street Journal and the independently-owned New York Times revealed in recent days that their networks were penetrated at the weekend, potentially by China-based hackers.