Popyrin won the first set against world No.69 Corentin Moutet but battled a left glute complaint and a cramping hamstring from midway through the second set, losing 4-6 6-3 6-4 6-4 at John Cain Arena.
It was a hammer blow for Popyrin, who was seeded for the first time and attempting to kick on from his bumper 2024, which included claiming a Masters 1000 title in Montreal and brilliantly upsetting Novak Djokovic to reach the fourth round at the US Open.
He also lost his first-round match in Brisbane.
"I'm going to have to take a few days, a week off, let it all sink in," Popyrin said.
"It's been a very disappointing month for me, come to Australia off the back of your best year, lose two matches is not something I kind of expected.
"Yeah, it's not been an easy month, that's for sure.Â
"We're going to have to take a step back, look at what went wrong, and just work from there.
"I haven't been in this position before, so I don't really know what I have to do, but it's all part of the experience."
The Australian relished his fighting spirit and a parochial crowd's energy - but little else.
"That's an experience and a memory I'll take with me, but the rest I'm just going to throw in the bin and forget, because it's not been great," he said.
Popyrin, 25, didn't have the answers for a familiar foe in Moutet, who he described as his "Achilles heel" since he was eight years old.
An injured Alexei Popyrin did it tough in his first-round loss to Frenchman Corentin Moutet. (Joel Carrett/AAP PHOTOS)
Popyrin, 25, saved nine break points as he won the first set.
But in the second, he took a medical timeout when trailing 3-2 on serve, receiving treatment on his left glute.
"I ran to a drop shot, felt a pull - a sharp pain in my glute, like lower backside glute. I have had it before, and I knew what it was," he said.
"It was really painful for me to push off my left leg and land on my left leg and do running forehands and stuff.
"It's something that I knew, I think it couldn't get worse, but yeah, I just had to dig deep and play through the pain."
Popyrin revealed he had been sick for the past four days and also dealt with an abdominal issue, but didn't want to use it as an excuse.
"Like, I take full responsibility with the way that I played, and, you know, it was shocking. It was really bad. And I know I can do better," he said.
Popyrin was hopeful his injury would recover within two-to-three weeks.
"But it's something you can play through, so it's just I have to decide whether or not I want to play through it and risk it getting worse or not," he said.
Moutet levelled the match then won the third set, despite a Popyrin fightback.
The Frenchman then broke Popyrin early in the fourth and while the Sydneysider got the set back on serve, he was broken again at 4-4.
Popyrin saved two match points before Moutet claimed a victory that earns him a second round tie with American qualifier Mitchell Krueger, who also beat an Australian, Rinky Hijikata.
