The Italian Open was only Serena Williams’ fourth tournament of the season.
Her latest withdrawal means she will head to the French Open having played just eight full matches in 2019.
The 37-year-old was due to play sister Venus in an eagerly anticipated second-round cash in Rome.
Venus received a walkover into the third round.
Why did Serena Williams withdraw from Italian Open?
Williams has withdrawn from the Italian Open due to a knee injury.
She said: “I must withdraw from the Italian Open due to pain in my left knee.
“I will miss the fans and competition at one of my favourite tournaments.”
Williams took nearly two months off after withdrawing ahead of her second match in Miami in March, also with a knee problem.
Earlier that month, she retired mid-match at Indian Wells because of illness.
Will Serena Williams play at the French Open?
Despite her latest injury setback, Williams is confident of being fit to feature in Paris.
She said: “I’ll be concentrating on rehab and look forward to seeing you all at the French Open and next year in Rome.”
Williams, currently ranked 11th, made her grand slam return in Paris 12 months ago following the birth of her daughter.
She reached the fourth round before withdrawing ahead of a much-anticipated clash with Maria Sharapova because of a shoulder problem.
The French Open begins on May 26.
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