Stosur became the first Australian woman to win a Grand Slam title since Evonne Goolagong Cawley won at Wimbledon in 1980, doing so against a future Hall-of-Famer who had not lost a set leading up to the final.
"I can't actually believe I won this tournament," Stosur said. "To go out there and play the way I did is obviously just an unbelievable feeling. You always hope and want to be able to do that, but to actually to that is unbelievable."
A sluggish Williams, who had ousted top-seeded Caroline Wozniacki less than 17 hours before the start of Sunday's showdown, had her serve broken an uncharacteristic five times, including in the final game to fall, 6-2, 6-3.
The quick turnover affected the American early on, as she lost her second service game and improbably dropped the final 12 points of the opening set.
Stosur, seeded ninth, also won the first point of the second set and took the game in controversial fashion. Williams, trailing 30-40, hit a shot that was well out of Stosur's reach and yelled in excitement as the ball touched the top of her opponent's racket.
The chair umpire ruled Williams had interfered in a potential return, and subsequently awarded the point to Stosur.
Williams, who famously lost her composure in an Open semifinal match against Kim Clijsters two years ago, gained life from the perceived slight and broke Stosur's serve for the first and only time in the match.
Stosur, undaunted, stayed the course while the crowd tried to will Williams back into the match. The manufactured energy was short-lived, however, and Stosur ended things rather quickly to become the third straight first-time winner at a major.
With the set even at 3-3, Stosur converted a break point before holding serve to put Williams' back to the wall. Williams saved two match points to extend the match, but Stosur hit a pair of forehand winners in the deuce to seal the biggest win of her career.
Williams finished with 25 unforced errors to Stosur's 12 and converted just 52 percent of her first serves.
"I guess my serve just wasn't working for me today," Williams said. "I think sometimes a lot of people were putting me as the favorite, and I definitely was trying not to put myself as the favorite. ... It's anyone's game, as you can see [by the] result today."
Stosur, the first Australian woman to reach the U.S. Open final since it moved to Flushing Meadows in 1978, had appeared in only one previous Grand Slam final, losing to Francesca Schiavone in the 2010 French Open.
Williams, seeded 28th in this year's tournament mostly because she missed an extended amount of time with injuries, was vying for her fourth U.S. Open title and first since 2008.
Stosur is expected to rise to No. 7 in the new WTA rankings, while Williams, who fell to 13-4 in Grand Slam finals, will vault up to No. 14.