Kvitova beat Greet Minnen in her first-round match Sunday in Paris. She was scheduled to face Elena Vesnina in the second round Wednesday at Roland Garros. She said she sustained the injury during Sunday's post-match media availability.
"It is with great disappointment that I announce my withdrawal from Roland Garros," Kvitova wrote on social media.
"During my post-match press requirements on Sunday, I fell and hurt my ankle. Unfortunately, after an MRI and much discussion with my team, I have made the tough decision that it would be unwise to play on.
"It's incredibly bad luck, but I will stay strong and do my best to recover in time for the grass-court season."
Kvitova's withdrawal comes one day after No. 2 Naomi Osaka pulled out of the tournament Monday to address mental health concerns. Osaka said last week she would not participate in the event's mandatory news conferences.
World No. 7 Bianca Andreescu and No. 13 Garbine Muguruza each lost their first-round match Monday in Paris, which means only nine of the Top 13 women's players still are active in the singles circuit of the Grand Slam.
Vesnina automatically advanced to the next round because of Kvitova's withdrawal. She faces Elena Rybakina or Nao Hibino in the third round this week in Paris.