The 33-year-old, who has spoken openly of retiring, returned to tennis following a two-and-a-half-year injury absence for the Argentine Open but lost in the first round on Tuesday.
"Unfortunately, Juan Martin del Potro will not play the Rio Open," tournament organizers said on Twitter, a day before the opening match.
"It was always a dream to have him with us, and we nearly did. We wish him much success in the future. Please know that the tournament's doors will always be open to you."
Del Potro said he has been "living a nightmare" since he fractured his kneecap in June 2019 at Queen's in London.
The former US Open champ lost in straight sets (6-1, 6-3) to compatriot Federico Delbonis in Buenos Aires on Tuesday.
He said before that tournament that his comeback was "more a farewell than a return."
"Possibly we may not meet again," an emotional Del Potro told fans after the match.
"Today I gave everything I had, until the last point. I'm happy because my last game was probably on a tennis court in front of all of you, and not in a press conference."
However, he added at the time that he wanted to "leave the window open" for Rio de Janeiro.
Del Potro beat Roger Federer in the 2009 US Open final, the high point of a career that has seen him win 22 titles, the 2016 Davis Cup for Argentina, and Olympic silver and bronze.
But he has often struggled with injury.
Once world number three, his ranking has gone to 753 since his knee injury, which required four surgeries.
