The announcement came after the Mail on Sunday newspaper reported that Fiennes had had an affair with a Romanian singer.
"Ms. Annis confirms today that she and Ralph Fiennes are to separate," said a statement issued Tuesday by her law firm, Schillings.
Fiennes, 43, and 61-year-old Annis met while starring in a 1995 production of "Hamlet," in which Fiennes played the title role and Annis his mother. Fiennes was married to "ER" star Alex Kingston at the time, while Annis had three children with photographer Patrick Wiseman.
Schillings said Annis had begun legal action against The Daily Mail newspaper for defamation and invasion of privacy over a story claiming she had forgiven Fiennes for his affair.
Fiennes is nominated for best actor at next week's British Academy Film Awards for his portrayal of a diffident diplomat in the spy thriller "The Constant Gardener."
The two-time Academy Award nominee — for "Schindler's List" and "The English Patient" — is known to young movie-goers as evil Lord Voldemort in " Harry Potter and the Goblet of Fire."
Annis, a leading stage actress, has appeared in films including "Dune" and "The Libertine."