On a rainy night in New York, Osaka was beaten in straight sets by American Danielle Collins 7-6(5) 6-3, while defending champion Emma Raducanu lost to Alizé Cornet.
19-year-old Raducanu stunned the world when she won her first Grand Slam at Flushing Meadows last year, but has struggled to find similar form since.
The experience and intensity of the seasoned Cornet proved too much for her as she was defeated 6-3, 6-3 in straight sets.
“This obviously hurts a bit because it’s my favorite tournament and there’s been a lot of emotion over the past year,” she told reporters after the loss.
“I’m proud that in every game, every day, I push myself because I know I’m striving to do the best I can.”
Raducanu will fall down the rankings after a frustrating year plagued by injuries and changes in their coaching staff.
The youngster has struggled to come to terms with life on the full WTA Tour but has shown flashes of quality that secured her maiden Grand Slam title just 12 months ago.
Cornet has simply been in better form and is now familiar with big results in the Slams, having previously beaten the likes of Iga Swiatek at Wimbledon.
The French now holds the Open-era record for most consecutive Grand Slam appearances, having appeared in the previous 63.
“I think I manage my emotions better,” Cornet told reporters after the game. “I think I’m getting old – more mature. This shows in my results. I’m 32 – better late than never.”
“She definitely ratted me out last time”
Osaka endured a similar experience to Raducanu but this time at the hands of the talented Collins.
The world No. 19 had never won a set against Osaka in all their previous three encounters, but this time she overtook the four-time Grand Slam champion.
“If you lose to someone three times, you get a lot of information about what you can do better,” Collins told reporters.
“Being the player that Naomi is, she definitely cheered me on last time out. I just had to learn from it and make adjustments, and I think I did.”
Osaka, a two-time US Open champion, was not seeded at this year’s event and is currently ranked 44th in the world.
Struggling with momentum and form, she was probably an outsider coming into the first round but put on an impressive performance despite the loss.
Collins now plays Spanish qualifier Cristina Bucșa in the next round, while Cornet meets Katerina Siniaková.