The 23-year-old had hoped to play a starring role at the Games on and off court, but lighting the Olympic flame at the opening ceremony will be the highlight.
Osaka had done well in her first two matches, especially against Viktorija Golubic on Monday, but former Roland Garros finalist Vondrousova took a step forward and the young Czech put in a great performance under the roof of Ariake Tennis Park to win 6-1 6-4.
Vondrousova mixed her trademark shots with big serves and powerful groundstrokes and defended brilliantly when Osaka pitched their game in the second set.
The four-time Grand Slam champion hasn’t moved as well as she could, possibly a legacy of the eight-week break she took for mental health reasons ahead of this tournament.
Osaka had won 25 of their last 26 matches on hard courts, winning Grand Slam titles at the US Open and Australian Open and, especially given the early defeat of Wimbledon champion Ashleigh Barty, the stage seemed set for her to claim arguably the biggest title in the mall.
But instead, 22-year-old Vondrousova becomes the first player to qualify for the quarter-finals, where she will face Spain’s Paula Badosa or Argentina’s Nadia Podoroska.
Osaka, who had hoped to become the first Japanese female tennis player to win Olympic gold, had no response from Vondrousova in the first set.
She responded well early in the second with a break of serve but was quickly pegged and, although she saved two match points at 5-4, another followed and Osaka sent a wide backhand.
Osaka left Ariake Tennis Park minutes after their loss, refusing to go through the mixed zone and conduct interviews.
She will probably be next at the US Open.
Speaking ahead of the tournament, she explained why she chose to represent Japan and not the United States, explaining the racism she was subjected to for her decision.
Osaka said, “I have been playing for the Japanese flag since I was 14. It was not even a secret that I will be playing for Japan for the Olympics.
“So I don’t choose America and all of a sudden people are like, ‘Your black card is revoked. And it’s like the African American isn’t the only black man, you know?
“I don’t know, I have the impression that people really don’t know the difference between nationality and race because there are a lot of blacks in Brazil, but they are Brazilian”,