Twenty-four hours after Kvitova had crashed out at the hands of Alexandra Dulgheru, Chinese star Li went down 6-2 7-5 to her Romanian compatriot, Simona Halep.
A surprise winner on the Paris clay back in June, Li had proved her hardcourt credentials at the start of the season when she made the final of the Australian Open.
However, Flushing Meadows did not prove to be to her liking.
Halep dominated the first set and when she moved a break up in the second, the Louis Armstrong Stadium crowd sensed a shock.
The sixth seed managed to get back on level terms but then lost her serve again at 5-5 before Halep served out.
With Australian Open champion Kim Clijsters, who also won the US Open in 2009 and 2010, absent this year through injury, it is the first time since 1971 that no reigning grand slam champion has made it through to the second round.
Seventh seed Francesca Schiavone, last year's French Open champion, had to fight hard to avoid a similar upset against Galina Voskoboeva, of Kazakhstan.
Schiavone started well enough but went off the boil in the second set before scrapping to a 6-3 1-6 6-4 victory.
A total of 16 double faults did not help the Italian's cause and she will need to improve to stand any chance of contending in New York.
There was also a scare for Ana Ivanovic, the former French Open champion seeded 16 at this event.
She had to have her blood pressure taken during her clash with Ksenia Pervak but recovered to post a 6-4 6-2 win.
Ivanovic, who was a break down early on in the match, has never been beyond last 16 in New York.
Another Grand Slam winner of years gone by, Svetlana Kuznetsova, had no such problems as she eased past Sara Errani.
The 2004 US Open champ was a 7-5 6-1 victor over the Italian.