The world number one, who is being chased in the rankings by German Angelique Kerber, took a free entry only last week and was seeded atop the draw.
But the 34-year-old American reversed course abruptly and withdrew from the tournament on Monday.
“I’m very disappointed I’m not able to compete as I was really looking forward to defending my title,” Williams said.
“My shoulder inflammation continues to be a challenge, but I am anxious to return to the court as soon as possible.”
The absence of Williams now opens the door to Kerber to make a run at the WTA top ranking. Williams has been No. 1 for 183 straight weeks and 306 overall.
But Kerber can become number one if she wins the Cincinnati title.
Williams took a wild card spot to get into the draw. She will be replaced by lucky loser Misaki Doi of Japan.
Doi moved into the second round and will face American Christina McHale, who beat Zheng Saisai of China 6-2, 6-2.
Australian Daria Gavrilova defeated Caroline Garcia of France 7-5, 6-3; Latvian Jelena Ostapenko stopped Anna Karolina Schmiedlova of Slovakia 1-6, 7-6 (8/6), 7-6 (7/5).
Ukrainian Lesia Tsurenko defeated China’s Zhang Shiau 6-3, 6-3 while American CoCo Vandeweghe put out Italian Sara Errani 4-6, 6-3, 6-4. Croatian Donna Vekic shocked newly married former world number one Ana Ivanovic 6-4, 6-2.
In men’s play on a day interrupted by rain, France’s Julien Benneteau earned the first upset win of the week as he put out Spanish 10th seed David Ferrer 6-4, 2-6, 6-4.
Injury comeback hope Kevin Anderson of South Africa reached the second round when Alexandr Dolgopolov — a semi-finalist here last year from a qualifying start — was unable to carry on after losing the first set 7-5.
Uruguay’s Pablo Cuevas beat Californian Sam Querrey 4-6, 7-6 (7/5), 6-3, finishing just before the rain arrived.
Marin Cilic reached the second round by beating Serb Viktor Troicki 6-3, 6-4, while Atlanta tournament champion Nick Kyrgios saw off Frenchman Lucas Pouille 6-2, 7-5.
Bulgarian Grigor Dimitrov shrugged off a first-round Olympic loss to defeat Gilles Simon 6-1, 6-3 while US teenager Reilly Opelka put out Jeremy Chardy 3-6, 7-5, 7-6 (11/9).
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