The four-time Italian Open champion said an injury to her left knee had forced her to withdraw from the Rome event.
“I will miss the fans and competition at one of my favorite tournaments," said Serena Williams. "I’ll be concentrating on rehab and look forward to seeing you all at the French Open and next year in Rome.”
On Monday, Williams beat Sweden's Rebecca Peterson in straight sets to set up the second round meeting with her sister Venus.
The Italian Open was Serena Williams' first tournament since she pulled out of the Miami Open in March just after dispatching Peterson.
Venus Williams, the 1999 winner in Rome, will play either the unseeded Briton Johanna Konta or the seventh seeded American Sloane Stephens.