Safarova, the 13th seed, won 7-6 (7/3), 6-4 to progress to a first ever quarter-final at Roland Garros, where she will face Garbine Muguruza after the Spaniard saw off Flavia Pennetta.
Sharapova, who also won the clay-court Grand Slam event in 2012 and was the beaten finalist in 2013, had been forced to wait until Monday morning for her clash with Safarova, which had been postponed on Sunday due to the rain in the French capital.
Sharapova was broken in her second service game, and although she soon broke back, Safarova claimed the opening set in the tiebreak on Court Philippe Chatrier.
Breaks were again exchanged in the second set but Sharapova found herself serving to stay in the match at 5-4 down, and while she saved one match point she could not save the second that came her opponent's way.
"It was a tough day at the office."
The Czech had not won any of her last four meetings with Sharapova since triumphing in their first ever clash in Madrid in 2010 and will now face Muguruza on Tuesday after the Spanish 21st seed breezed past Pennetta.
Muguruza won 6-3, 6-4 on Court Suzanne Lenglen to reach the last eight at Roland Garros for the second year in succession.
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