The U.S. Tennis Association announced the withdrawal of the 2006 champion, who has played only one match on tour since her second-round loss at Wimbledon in June.
Sharapova originally was seeded third at the U.S. Open. The USTA said 2012 Wimbledon runner-up Agnieszka Radwanksa would shift from No. 4 to No. 3, and all other seeded players below her would move up a spot, too.
"I just wanted to let you know that withdrawing from the U.S. Open has been a really tough decision to make. I have done everything I could since Wimbledon to get myself ready but it just wasn't enough time," read a message posted on Sharapova's Facebook page on Wednesday. "I have done many tests, received several opinions and it all comes down to taking the proper amount of time to heal my shoulder injury properly."
The USTA said Anastasia Pavlyuchenkova would become the No. 32 seed.
The draw for the year's last Grand Slam tournament is Thursday. Play begins Monday.
Sharapova's surprising exit caps a tumultuous couple of weeks for the four-time major winner and former No. 1-ranked player.
She was sidelined by a hip injury after Wimbledon, then hired Jimmy Connors as her coach - an arrangement that lasted all of one match, a loss.
Sharapova last skipped the U.S. Open in 2008, when she was off the tour for about 10 months because of surgery on her right shoulder.
She won her first major title since that operation at last year's French Open, completing a career Grand Slam.
Earlier Wednesday, former top-10 player Mardy Fish withdrew from the U.S. Open, citing personal reasons.