"American Serena Williams, who has not fully recovered from health problems - pulmonary embolism, foot injury - has pulled out," organisers said in a statement.
Serena, who has not played since last July, has dropped to 17th in the WTA rankings.
She won the French Open in 2002.
Her sister Venus is a major doubt for the May 22-June 5 tournament, having not played since January because of a hip injury.
Organisers also confirmed that former world number one Dinara Safina, who is taking an indefinite break from the sport, and Argentine Davis Nalbandian had pulled out of the tournament.
-ReutersTags: sport, tennis, france