For the third time in three round Osaka lost the opening set, but this time she was unable to turn things around.
"I just feel like there has been a weight on me, kind of", said Osaka, who was seeded No. 1 at a major tournament for the first time.
Osaka has yet to reach the fourth round of the French Open despite her career success, while for Siniakova it marks the first time she has reached this stage of any Grand Slam competition.
Osaka dropped the first set for the third successive match at the tournament and squandered seven break points in all.
Only four of the women's top 10 seeds are left in the tournament among them are three-time victor Serena Williams and defending champion Simona Halep.
"It's fantastic, I can't believe it, I am so happy now", said Siniakova. Her form was not that good after Melbourne thanks to some injuries and she wasn't among the favorites in Paris, barely surviving Anna Karolina Schmiedlova in the first round. She was 0 for 4 on break chances when Siniakova served out that set, then never managed to earn one in the second.
Labour will be complicit in a no-deal Brexit, says Alastair Campbell
It has been four years of rebuilding for the Lib Dems as they joined a hideously unpopular coalition with the Tories in 2010. YouGov surveyed 1,736 British adults on May 28-29.
She wound up with a hard-to-believe 38 unforced errors; Siniakova made only 13.
Siniakova, considered to be the one of the world's best doubles players, will now face 14th seed Madison Keys for a place in the quarter-finals and afterwards spoke of her joy at upsetting the odds against the world's best player.
At least it will release the sense of pressure that was building up through her run of 16 consecutive grand-slam victories.
Siniakova took control in the second set, converting the first of two break points to go 3-2 up.
Serving for an impressive victory, Katerina held at 15 to start a huge celebration after more mistakes from world no. 1 who played way beyond her level, unable to pull the third comeback of the week and stay on the title trail.