Osaka, who lit the Olympic cauldron during the opening ceremony, struggled in an error-strewn display under the centre court roof at Ariake Tennis Park and was knocked out in 68 minutes.
She made a dreadful start by dropping serve in the opening game. The second seed was broken twice more as 2019 French Open runner-up Vondrousova raced away with the first set.
Osaka broke in the second set but relinquished the early advantage with a double fault that allowed Vondrousova to level at two games apiece.
The 23-year-old four-time Grand Slam winner grappled with inconsistency, and even when given a sniff of regaining the initiative she had no response to Vondrousova's array of crafty drop shots.
Osaka saved two match points as she served to stay alive at 4-5 but Vondrousova converted at the third time of asking as the Japanese star smacked a backhand wide.
The defeat comes after a turbulent few months for Osaka, who first refused to attend press conferences at the French Open and then said she was taking time out from tennis as she battled depression and anxiety.
She missed Wimbledon but returned for the Olympic tournament and had seemed back on her game in the early rounds in Tokyo.
Vondrousova, ranked 42nd, will go on to face Spain's Paula Badosa or Nadia Podoroska of Argentina.