Pliskova -- a finalist at the 2016 U.S. Open -- failed to convert two set points in the second set and lost 6-1, 7-6 (2) to France's Caroline Garcia. It was the French player's fourth victory over Pliskova in their seven total meetings.
It marked the first time since 2008 that the top-seeded woman lost in the second round at Flushing Meadows.
"Sometimes the score can say it's really like there is a top player and then there is a bad one, but it's not really like this," Garcia said. "It's closer than it looks most of the time and a few points can make the switch. So I had to stay focused.
"I knew maybe she was going to come back. She was not going to give me the match for sure so I had to be ready for everything. When she came back very well I had to stay calm and that was most important."
Meanwhile, No. 4 seed Naomi Osaka cruised to a straight-sets win over Camila Giorgi in their second-round match. Osaka earned the 6-1, 6-2 victory in an hour and 10 minutes to advance to the third round.
Unseeded Americans Ann Li and Shelby Rogers also pulled off upset wins Wednesday. The 20-year-old Li defeated No. 13 seed Alison Riske 6-0, 6-3, while Rogers beat 11th-seeded Elena Rybakina 7-5, 6-1.
No. 31 seed Anastasija Sevastova, who knocked off Cori "Coco" Gauff in the first round, lost to unseeded Marta Kostyuk in another second-round upset.
Earlier Wednesday, 17th-seeded Angelique Kerber advanced to the third round with a 6-3, 7-6 (6) victory over Anna-Lena Friedsam. No. 6 seed Petra Kvitova also moved into the next round with her 7-6 (3), 6-2 triumph over Kateryna Kozlova.
On the men's side of the tournament, Novak Djokovic rallied past Kyle Edmund in Wednesday's second round in four sets. The top-seeded Serbian fell behind when he lost a tiebreaker for the first time this year but took the next three sets for a 6-7 (5), 6-3, 6-4, 6-2 win.
Djokovic -- seeking his fourth U.S. Open title -- had been 10-0 in tiebreakers this year. He extended his winning streak, which dates back to last season, to 28 matches overall.
Djokovic hit 16 aces and lost only 12 points on his first serve.
"I'm actually glad I did drop a set and got tested the way I did today against Kyle. I expected it to be a tough, tough task," Djokovic said. "I'm really glad [about] having an early, tough match in the tournament. It kind of serves me better, I think, for the rest of the tournament."
Djokovic, who has won five of the last seven Grand Slam tournaments, will face No. 28 Jan-Lennard Struff in the third round.
Earlier Wednesday, Alexander Zverev defeated 19-year-old American Brandon Nakashima 7-5, 6-7 (8), 6-3, 6-1. The fifth-seeded German overcame 10 double faults, hit 24 aces and won every service game.
No. 7 seed David Goffin also reached the next round with his win over Lloyd Harris.