On a breezy day with tricky hole locations at Blackwolf Run where only four other sub-par scores were recorded, Choi put herself in prime position to win her first major title.
The 24-year-old Korean, who has triumphed five times on the LPGA Tour, seized control of the tournament in masterly fashion with eight birdies and a sole bogey to finish at eight-under 208.
"I hit a lot of good shots today, my 14 clubs worked very well," a beaming Choi told reporters after totaling 26 putts on fast-running greens.
"I had a birdie on the first hole and a birdie on the second hole, then I got some good vibes from there, good confidence from there. It was really fun out there.
"I couldn't believe how I got eight birdies today, but I did," she grinned. "I'm very happy, I'm very satisfied and I'm very excited."
Choi's 65 was the 11th carded at a US Women's Open where only four lower rounds have ever been posted.
Compatriot Amy Yang was alone in second after carding a 69, the next-best score of the day, with American teenager Lexi Thompson (72), Japan's Mika Miyazato (73) and Germany's Sandra Gal (74) a further stroke back at one-under 215.
Overnight leader Suzann Pettersen of Norway endured a difficult day, battling to a 78 that included a pair of double-bogeys to finish a distant nine strokes off the pace.
Pettersen, who had led by one shot after the second round, briefly doubled her advantage when she birdied the par-five second.
However, she fell back into a tie for the lead with the first of her double-bogeys, at the par-four third, before Choi took control with a sizzling birdie display.
The petite Korean scorched the front nine in four-under 32 and then picked up further shots at the 10th, 11th and 12th to charge five shots clear at eight under.
Though she ran up a three-putt bogey at the tricky par-three 13th, Choi recovered with another birdie at the 17th to stretch her lead to six.
Since turning professional in late 2004, Choi has established herself as one of the leading players on the LPGA Tour.
In 2010, she won twice on the US circuit before ending the year by clinching the money list title and also the prestigious Vare Trophy for the lowest scoring average, with 69.8.
Choi has yet to win a major title, but this week offers a golden opportunity to change that.
"It will be a lot of pressure tomorrow, but I know what I have to do and I know what I can control," she said of Sunday's final round. "I cannot control winning. I just do my best.
"I have confidence and this is a good opportunity to be winning a US Open. So I just hope to get a good warm-up tomorrow morning, go out there with my caddie and have fun."
Pettersen, who won her first major title at the 2007 LPGA Championship, was dumbfounded by Choi's display on Saturday in tough conditions.
"Today wasn't easy. Today was kind of the biggest test," the 31-year-old Norwegian said. "Na Yeon played phenomenal golf. I couldn't believe the scores I saw, to be honest."
Asked how she viewed her own task on Sunday, Pettersen replied: "There's birdies out there. I think the wind is going to be a little bit less tomorrow from what I've seen.
"So if you get off to a hot start, hopefully put a number down early in the clubhouse, who knows?"
Taiwan's world number one Yani Tseng, who has claimed five of the last 10 Major titles, emulated Pettersen by carding a 78 on Saturday to lie a distant 16 strokes off the pace.
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