Williams' 73-minute masterclass on Centre Court clinched a 6-3, 7-5 victory that made her the most senior player to reach the semi-finals since Martina Navratilova in 1994.
The American star, who reached the last of her eight Wimbledon finals in 2009, faces Britain's Johanna Konta or Romania's Simona Halep in the last four on Thursday.
World number 11 Williams, beaten in this year's Australian Open final, was knocked out in the 2016 semi-finals and is chasing a first major title since winning Wimbledon in 2008.
She will climb back into the top 10 thanks to her Wimbledon run, but breaking Serena's record to become the tournament's oldest champion in the Open era is her sole focus.
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Twenty years after making her Wimbledon debut, Venus was playing in her 100th singles match at the All England Club, while Ostapenko was in only her eighth.
That gulf in experience was immediately apparent when Venus broke in the second game and cruised through the first set in serene fashion.
But Ostapenko, 20, was the youngest player left in the tournament and Venus has scythed through the draw by dispatching a series of opponents almost half her age, many of whom idolised the seven-time Grand Slam champion when they were growing up.
Williams was just too canny for the fresh-faced Ostapenko, manoeuvering her out of position time and again before delivering the kill shot.
After breaking in the third game of the second set, Williams moved into a 3-1 lead, then endured her only wobble as Ostapenko broke back.
Muguruza powered into her second Wimbledon semi-final in the last three years with an emphatic 6-3, 6-4 win over Russian seventh seed Kuznetsova.
Since winning her maiden Grand Slam title at the French Open last year, Muguruza has struggled to return to the top and this is her first major semi-final since that Roland Garros triumph.
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Muguruza, beaten by Serena in the 2015 Wimbledon final, will face Coco Vandeweghe or Magdalena Rybarikova in the last four.
The 23-year-old is being coached by Conchita Martinez, who became the only Spanish woman to win Wimbledon in 1994, and the lessons appear to have done the trick.
"It seems far away since I last made the final here. I'm a completely different player.
"It means a lot to make the semi-finals again, my breakthrough was here."
Following Venus onto Centre Court is Johanna Konta, who takes on Simona Halep bidding to become the first British woman to reach the semi-finals since Virginia Wade in 1978.
Not since Jo Durie in 1984 has Britain had a female representative in the last eight and Konta, hoping to become the first home winner of the Wimbledon women's title since Wade in 1977, has added incentive to go a step further against Halep after their stormy Fed Cup encounter earlier this year.
Wade is among those taking a seat in the Royal Box for the hotly-anticipated grudge match.
Halep has extra motivation of her own as a victory over Konta will guarantee she takes over from Angelique Kerber as the new world number one.