The 36-year-old Frenchman played only eight minutes Wednesday before completing a first-round loss to Christophe Rochus of Belgium 6-3, 6-1, 3-6, 6-4. The match had been suspended Tuesday.
"When I started my career on court No. 10 in 1989, I did not imagine at all that I would hold the microphone in my hands 20 years later in front of you," Santoro said to the crowd at Court Suzanne Lenglen. "Those were extraordinary and fantastic years that I will never forget."
Santoro has played in a record 67 Grand Slam tournaments, making the fourth round three times - at the French Open in 1991 and 2001 and at the Australian Open in 1999.
"Twenty years. That counts for something in a lifetime," Santoro said. "It has been a long road, a fantastic career. I had a lot of fun and learned a lot."
Santoro and Rochus started their match Tuesday, but it was suspended by darkness with the Belgian leading 5-3 in the fourth set. The pair came back out onto the court after Dinara Safina's easy win and finished the match quickly.
"I'm saying to myself that the story is over, that a page is being turned," Santoro said. "I will no longer be on court next year. But I think that it is time to go."
Santoro plans to play other tournaments before retiring at the end of the year.
ALSO ON THIS STORY Discuss: Sound off on tennis at Newsvine.com
Besides tying Francois Jauffret's record for appearances at the French Open, Santoro also holds the record for the longest match at the Paris tournament in the Open era.
"That was in 2004. A match lasting 6 hours, 33 minutes on this same court," said Santoro, who beat Arnaud Clement in the first round that year 6-4, 6-3, 6-7 (5), 3-6, 16-14. "Quite a few records over the long haul: The longest match, the greatest number of Grand Slam appearances. Those are figures that are going to my head. I couldn't imagine that when I started playing."
Also on NBCSports.com
NBA: NBA could eliminate ref miscues in an instant
NHL: Wings set up Cup rematch with Pens | Preview
Celizic: Mad Manning signals bad news in Indy
Boxing: Is Tyson doomed to tragic life? | Images
Austin: Sharapova shows fine form at French Open
