The veteran has booked a semis berth against Marie Bouzkova.
Meanwhile, Osaka moved to the number one spot in the WTA rankings, despite the defeat.
In men's section, defending champion Rafael Nadal also moved to the semis.
Here are the details.
Williams capped off a stunning 6-3, 6-4 win to overpower Osaka.
Williams, who is yet to drop a set this week said, "It's definitely not where I want to be, but I'm getting there."
Meanwhile, her opponent Bouzkova, who is ranked 91st in the world, progressed to the semis after her opponent Simona Halep retired hurt after losing the first set 4-6.
Osaka saw her number one status get confirmed after Karolina Pliskova lost 0-6, 6-2, 4-6 to Canadian teenager Bianca Andreescu.
Pliskova, who was a former world number one for eight weeks in 2017, needed to win the tournament to regain the top spot.
Also, Ashleigh Barty replaced Osaka at the top in June.
Her campaign ended with a loss in round two.
Our #RC19 semi-finals are set!— Rogers Cup (@rogerscup) August 10, 2019
Bianca Andreescu 🇨🇦 vs. Sofia Kenin 🇺🇸
 Serena Williams 🇺🇸 vs. Marie Bouzkova 🇨🇿
Come be part a part of it with us ➡️ https://t.co/2CZdD5WJrk pic.twitter.com/vq5tHudULX
In the men's section, defending champion Nadal beat Fabio Fognini to move into the semi-finals of the Rogers Cup in Montreal.
The current world number two is seeking a fifth Rogers Cup title.
He started slowly before dispatching the Italian 2-6, 6-1, 6-2 in a three-set affair.
The Spaniard will next face Gael Monfils or Roberto Bautista Agut in the semis.
“I asked about what’s going on in his foot because I saw him asking for treatment. Something I experienced before. I just tried to tell him my experience (...) and how I went through that process that he’s having.”— Coupe Rogers (@CoupeRogers) August 10, 2019
@RafaelNadal - classiest man in the sport 🙏#CoupeRogers pic.twitter.com/5ayW9Iae4y
The other key results saw eighth-seeded Medvedev breeze past Austrian second seed Dominic Thiem 6-3, 6-1. Meanwhile, Khachanov overcame German number three seed Alexander Zverev 6-3, 6-3 in a relatively one-sided affair.
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