Williams insists she was trying not to make the same mistake as last year and let an injury affect her chances at Roland Garros, or even Wimbledon.
Williams, who exited the Madrid Open in the semi-finals last week, said the decision was made after lengthy consideration.
“We have to make the right decisions for the future, not for now,” Williams said. “You know I hate, hate quitting, and this isn’t quitting, it’s just making a good decision.
“I was really injured last year, actually, and ended up taking like five days off before Paris and practicing just a day or two before the tournament started,” added Williams, who lost in the second round.
“And entering a Grand Slam, you never want to enter it like that, especially as defending champion.”
Williams’ withdrawl meant that American qualifier Christina McHale made the last eight.
Also, two-time champions Maria Sharapova came back from a 3-1 first-set deficit to beat Bojana Jovanovski 6-3 6-3.
Rome winner in 2011 and 2012, the Russian has taken up the favourite’s mantle after Williams’ withdrawl..
Fourth seed Petra Kvitova proved too powerful for former world No 1 Jelena Jankovic, hitting 23 winners, with only 19 unforced errors.
Second-seeded Simona Halep routed 1999 Rome champion Venus Williams 6-2 6-1 and Carla Suarez Navarro defeated Eugenie Bouchard 6-7 (6-2) 7-5 7-6 (9-7).