The 32-year-old Czech reached the 2015 final where she was beaten in three sets by Serena Williams, and also won five Grand Slam doubles titles with Bethanie Mattek-Sands.
The American was missing this week with a knee injury, so Safarova played her final match alongside Slovakia's Dominika Cibulkova, losing 6-4, 6-0 to Sofia Kenin and Andrea Petkovic.
"Even after so many years, I still enjoy (it) when I'm on the court," said Safarova.
"I'm sure I will miss the competition a little bit. But honestly, I've been through great results on great courts, played in front of great crowds, and it's been great.
"It's been enough, and I'm really looking forward to starting a new chapter."
Safarova also won Olympic doubles bronze in 2016 with Barbora Strycova, with the pair stunning two-time defending champions Serena and Venus Williams in the first round.
But she said her final defeat by 23-time Grand Slam champion Serena remained the highlight of her career.
"There were many great matches. There were matches that hurt a lot, of course. You tend to remember those a little more for some reason.
"But I will definitely remember the finals here in Paris against Serena, (and) winning each of the Grand Slams next to Bethanie."
Safarova, who struggled with injuries and illness in the final two years of her career, reached a highest singles ranking of fifth and was the world number one in doubles.
Results from the fourth day of the French Open on Wednesday (x denotes seeding):
Stefanos Tsitsipas (GRE x6) bt Hugo Dellien (BOL) 4-6, 6-0, 6-3, 7-5
Filip Krajinovic (SRB) bt Roberto Carballes Baena (ESP) 6-4, 6-4, 6-7 (1/7), 3-6, 8-6
Stan Wawrinka (SUI x24) bt Christian Garin (CHI) 6-1, 6-4, 6-0
Grigor Dimitrov (BUL) bt Marin Cilic (CRO x11) 6-7 (3/7), 6-4, 4-6, 7-6 (7/2), 6-3
Nicolas Mahut (FRA) bt Philipp Kohlschreiber (GER) 6-3, 6-3, 6-3
Casper Ruud (NOR) bt Matteo Berrettini (ITA x29) 6-4, 7-5, 6-3
Roger Federer (SUI x3) bt Oscar Otte (GER) 6-4, 6-3, 6-4
Kei Nishikori (JPN x7) bt Jo-Wilfried Tsonga (FRA) 4-6, 6-4, 6-4, 6-4
Laslo Djere (SRB x31) bt Alexei Popyrin (AUS) 6-4, 7-6 (7/4), 6-4
Pablo Carreno Busta (ESP) bt Alex De Minaur (AUS x21) 6-3, 6-1, 6-1
Benoit Paire (FRA) bt Pierre-Hugues Herbert (FRA) 6-2, 6-2, 5-7, 6-7 (6/8), 11-9
Juan Ignacio Londero (ARG) bt Richard Gasquet (FRA) 6-2, 3-6, 6-3, 6-4
Corentin Moutet (FRA) bt Guido Pella (ARG x19) 6-3, 6-1, 2-6, 7-5
David Goffin (BEL x27) bt Miomir Kecmanovic (SRB) 6-2, 6-4, 6-3
Rafael Nadal (ESP x2) bt Yannick Maden (GER) 6-1, 6-2, 6-4
Sloane Stephens (USA x7) bt Sara Sorribes Tormo (ESP) 6-1, 7-6 (7/3)
Polona Hercog (SLO) bt Jennifer Brady (USA) 6-3, 6-7 (8/10), 6-4
Garbine Muguruza (ESP x19) bt Johanna Larsson (SWE) 6-4, 6-1
Elina Svitolina (UKR x9) bt Kateryna Kozlova (UKR) -- walkover
Donna Vekic (CRO x23) bt Rebecca Peterson (SWE) 1-6, 6-1, 6-2
Johanna Konta (GBR x26) bt Lauren Davis (USA) 6-3, 1-6, 6-3
Viktoria Kuzmova (SVK) bt Kiki Bertens (NED x4) 3-1 -- retired
Marketa Vondrousova (CZE) bt Anastasia Potapova (RUS) 6-4, 6-0
Carla Suarez Navarro (ESP x28) bt Shelby Rogers (USA) 6-1, 6-7 (4/7), 6-3
Elise Mertens (BEL x20) bt Diane Parry (FRA) 6-1, 6-3
Anastasija Sevastova (LAT x12) bt Mandy Minella (LUX) 6-2, 6-4
Veronika Kudermetova (RUS) bt Zarina Diyas (KAZ) 7-5, 6-1
Kaia Kanepi (EST) bt Shuai Zhang (CHN) 6-7 (4/7), 6-0, 7-5
Petra Martic (CRO x31) bt Kristina Mladenovic (FRA) 6-2, 6-1
Karolina Pliskova (CZE x2) bt Kristina Kucova (SVK) 6-2, 6-2