Osaka had hoped to become the first Japanese tennis player to win a gold medal.
The 23-year-old had hoped to play a starring role in the Games on the court as well as off it but lighting the Olympic flame at the opening ceremony will remain the high point.
Osaka had looked good in her first two matches, particularly against Viktorija Golubic on Monday, but former French Open finalist Vondrousova was a step up in level and the young Czech produced an excellent performance under the roof at the Ariake Tennis Park to win 6-1 6-4.
Vondrousova mixed her trademark drop shots with big serves and powerful groundstrokes and defended brilliantly when Osaka got her game going in the second set.
The four-time grand-slam champion did not move as well as she can, perhaps a legacy of the eight-week break she took for mental health reasons prior to this tournament.
Osaka said: “I definitely feel like there was a lot of pressure for this. I think it’s maybe because I haven’t played in the Olympics before and for the first year (ce) was a bit much. I think I’m glad with how I played, with taking that break that I had.
“I’ve taken long breaks before and I’ve managed to do well. I’m not saying that I did bad right now, but I do know that my expectations were a lot higher.
“I feel like my attitude wasn’t that great because I don’t really know how to cope with that pressure so that’s the best that I could have done in this situation.”
Osaka initially appeared to have left the site without talking to the media – in her social media post ahead of the French Open where she announced she would not be talking to the press she likened press conferences after losses to kicking someone when they are down.
pourtant, she returned to answer questions and admitted it was a painful defeat, en disant: “I’m disappointed in every loss, but I feel like this one sucks more than the others.”
Osaka had won 25 of her last 26 matches on hard courts, winning grand slam titles at the US Open and Australian Open, et, particularly given the early defeat for Wimbledon champion Ashleigh Barty the stage seemed set for her to claim arguably the biggest title of them all.
But instead it was 22-year-old Vondrousova who reached her first Olympic quarter-final, where she will take on either Paula Badosa of Spain or Argentina’s Nadia Podoroska.
Osaka, who had hoped to become the first Japonais tennis player to win Olympic gold, had no answer to Vondrousova in the first set.
She responded well at the start of the second with a break of serve but was quickly pegged back and, although she saved two match points at 5-4, another followed and Osaka sent a backhand wide.
She will now turn her attentions to the defence of her title in New York, although Osaka added: “I am a person who wings a lot of things. That is either a really good thing or a really bad thing.”
Stefanos Tsitsipas gained revenge for his Wimbledon loss to Frances Tiafoe by beating the American 6-3 6-4.