The 32-year-old Dutchman flew to the United States this week to consult surgeon Richard Steadman, who operated on the same knee in 2000 while the player was at PSV Eindhoven.
After tests and arthroscopic surgery, it was decided to operate again to try to repair damage to the external meniscus, the club said on its website.
"Taking all this into account, the estimated length of absence will be between six and nine months, depending on the clinical development," it added.
Van Nistelrooy damaged the knee during Real's 2-1 Champions League defeat at Juventus last month and missed the Primera Liga matches against Almeria and Athletic Bilbao.
He returned for the 2-0 home defeat by the Italians, but a scan appeared to show an aggravation of the injury that threatened to end his career eight years ago.
The length of his absence is a fresh blow to Real, who are struggling through a poor spell of form and are already missing key players including Dutch winger Arjen Robben, central defender Pepe and midfielder Ruben de la Red.
"It's very sad because Ruud is a very important player," striker Gonzalo Higuain said, adding that he hoped it did not spell the end of van Nistelrooy's career.
"We hope he will be back with us as soon as possible because he is a great player and a very good person," he told reporters.
Van Nistelrooy signed with Real at the end of the 2005-06 season from Manchester United for a fee reported by Spanish media to be 15 million euros ($18.7 million), helping the club to consecutive league titles in 2007 and 2008.
He was Real's top scorer in his first season with 25 league goals and equalled a club record of scoring in seven matches in succession. He netted 16 league goals last season and extended his contract to keep him in Madrid until 2010.
