The 34-year-old, who completed his career Grand Slam with victory at Roland-Garros in 2009, has been dealing with a series of injuries in 2016, including recovery from knee surgery following the Australian Open and a back injury in recent weeks.
Federer took to Philippe-Chatrier Court for a short practice session on Wednesday in the hope that he had made a return to full fitness in time for his 68th Grand Slam.
But the Swiss took to Facebook on Thursday to announce his decision to retire, ending his record run of consecutive Grand Slam appearances at 65.
"I regret to announce that I have made the decision not to play in this year’s French Open," Federer wrote.
"I have been making steady progress with my overall fitness, but I am still not 100% and feel I might be taking an unnecessary risk by playing in this event before I am really ready.
"This decision was not easy to make, but I took it to ensure I could play the remainder of the season and help to extend the rest of my career.
"I remain as motivated and excited as ever and my plan is to achieve the highest level of fitness before returning to the ATP World Tour for the upcoming grass court season.
"I am sorry for my fans in Paris but I very much look forward to returning to Roland-Garros in 2017."
Roland-Garros 2016 begins on Sunday 22 May. The draw ceremony will take place on Friday 20 May at 11.15am.
Well this was inevitable, sad that his Grand Slam appearance streak had to end in such a way.
But I would rather he be 100 % for Wimbledon, the Olympics and the U.S. Open in Sept. than having these continuous starts and stops throughout the rest of the season.
It will be strange not having him in the draw at all at a Grand Slam for the first time in so many years.
At least Martina Hingis & Sania Mirza are still going to play in hopes of capturing their "Santinaa Slam".
So that's something to look forward to.