Radwanska dropped just one game in a rapid first-round win over Sharon Fichman but world No 39 Peng was a deserved 6-3 6-4 winner.
The Chinese player looked like she might have missed her chance when two match points went begging, one on the Radwanska serve at 5-3 and the second on her own delivery.
Radwanska had a chance to make it 5-5 but missed it and Peng took her third match point with a backhand winner onto the line.
It was déjà vu for Radwanska, who suffered her only previous loss to Peng in the second round at Flushing Meadows four years ago.
Radwanska came into the tournament in a positive frame of mind after winning the title in Montreal earlier this month but she has never found the US Open a happy destination.
The Pole has reached at least the quarter-finals at every other grand slam but has not made it past the fourth round in New York.
Radwanska said: “It always is disappointing [to go out], especially in the first week of a grand slam. But I think here it’s not really a new thing for me unfortunately.
“I really want to know the answer. Then maybe I will find a way to play better here and just try another way to be in the second week.
“There is just something here that I can’t really pass those rounds. I have couple of years to try. Hopefully one day I will do it.”
Radwanska did not feel she gave a bad performance, adding: “Every game was pretty tight and a lot of rallies. But I think in the important moments she was just playing a little bit better.”
There was disappointment, too, for 21st seed Sloane Stephens, who from a set and 3-0 up against Johanna Larsson slumped to a 5-7 6-4 6-2 loss.
The American had balanced out poor performances in regular tour events with success at the slams but lost in the first round at Wimbledon and has underachieved again here.
There was a notable victory, meanwhile, for 17-year-old Belinda Bencic, who defeated 31st seed Kurumi Nara 6-4 4-6 6-1.
Meanwhile, Andy Murray will hope he can at last trust his body when he plays his second round match against Matthias Bachinger today.
The Scot arrived in New York in a positive frame of mind and declared himself in his best shape since winning Wimbledon last year following a training camp in Miami.
After 18 months of back problems followed by surgery and then the lengthy process of getting back to full fitness, it has been a long time since Murray felt at the peak of health on the court.
He thought that would be the case in his first-round match against Robin Haase on Monday, only for cramp to come on at the start of the third set and very nearly end his tournament there and then.
Murray dismissed nerves as a cause and has instead been focusing on what he ate and drank in the build-up to the match.
But it would not be surprising if Murray had been tense considering he has been unable to get back to his best form since back surgery almost a year ago.
Roger Federer endured his own back problems last season, although did not need surgery, and found his mind took longer to heal than his body.
Murray can empathise, saying: “I think if you have a physical issue that’s hampering you, it’s very easy for that to take over your thinking when you’re on the court.
“It’s frustrating to not be able to do the things that you want your body to do and that distracts you from what you should be doing on the court, which is trying to beat your opponent, not how you have to move to your backhand side or your forehand side or whatever the issue is.”
Although Murray insisted he does not need to take confidence from other players, seeing the way Federer has bounced back this season can surely be a source of comfort.
Having struggled throughout 2013, Federer has been in prime form this season, winning three ATP Tour titles and coming extremely close to an 18th grand slam title at Wimbledon before being pipped by Novak Djokovic.
Although Djokovic is the favourite to win at Flushing Meadows, it would certainly be no surprise if Federer lifted the trophy.