The top-ranked Williams has won three titles at Flushing Meadows, part of her 13 Grand Slam singles championships, the most among active women. Last year, she lost in the U.S. Open semifinals after a tirade at a line judge over a foot-fault call, an outburst that drew a record fine.
"It is with much frustration and deep sadness that I am having to pull out of the U.S. Open," Williams said in a statement released by her publicist.
She is the first No. 1 woman to miss the U.S. Open since the rankings began in 1975, according to the U.S. Tennis Association.
Williams said doctors advised her not to play so her foot can heal, and she called missing the tournament "one of the most devastating moments of my career."
The 28-year-old American reportedly was hurt by a broken glass at a restaurant while she was in Munich last month -- shortly after winning her fourth Wimbledon singles title on July 3, and before playing in an exhibition match against Kim Clijsters that drew a tennis-record crowd of 35,681 in Brussels on July 8.
Williams had surgery in Los Angeles on July 15. She already had pulled out of three hard-court tournaments she was scheduled to enter in preparation for the U.S. Open and also skipped playing World TeamTennis.
Andy Roddick, the 2003 U.S. Open champion, said Serena Williams' absence is a "fairly big-sized hit for the tournament."
Williams' withdrawal also means she won't team with older sister Venus to defend the doubles title they won in New York last year. Venus, who hasn't competed on tour since Wimbledon because of a bothersome left knee, is expected to play singles at Flushing Meadows, where play starts Aug. 30.