The 23-time grand slam winner returned earlier this year from a lengthy lay-off after the birth of her first child and still managed to make the Wimbledon final, which she lost to Angelique Kerber.
But Williams suffered the worst defeat of her career this week at the Silicon Valley Classic when she went down 6-1 6-0 to Johanna Konta and she will now miss next week's WTA Tour event in Montreal.
"Of course, we are disappointed that Serena will not be joining us. Fans were very much looking forward to seeing her in action," Rogers Cup tournament director, Eugene Lapierre, said via in a Tennis Canada statement.
"But beyond the disappointment, the tournament, as a whole, remains a high-level competition. The entire top 10 is here, along with 22 of the top 25. There are exciting matches in store from the outset."
German player Tatjana Maria will take Williams's place in the main draw and face Alize Cornet in the first round.