Pliskova, elevated to the head of the field in New York after a slew of high-ranking player withdrawals, never looked comfortable as she slipped to a 6-1, 7-6 (7/2) loss against a confident Garcia.
Garcia, the world number 50, will now face American 28th seed Jennifer Brady in the third round on Friday.
It was a miserable exit for Pliskova, the 28-year-old world number three from the Czech Republic, who was always struggling after a sluggish start.
Garcia raced into a 5-0 lead after scoring two service breaks before taking the first set.
Although Pliskova improved in the second, she was unable to find the crucial service break when it mattered.
In the tie break, Garcia raced into a 6-2 lead and then completed victory when Pliskova sent an unforced error into the net.