England's 41-game unbeaten run is over after they were stunned by a late rally from Canada, the Red Roses falling 24-21 in their WXV Global Series clash in Toronto.
Shoshanah Seumanutafa came off the bench to score the match-winning try in the 77th minute to hand England their first defeat since the 2022 World Cup final.
Following a draw in their previous clash last month in Exeter, Canada looked up for the challenge early as they opened the scoring through Savannah Bauder in the 15th minute.
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Quickfire tries to Maddie Feaunati and Zoe Harrison just before half-time put England ahead 14-5 at the break.
The points kept flowing as both teams crossed just after play resumed, but DaLeaka Menin's 63rd-minute try kept the door open for Canada.
A raucous home crowd exploded for Seumanutafa's match-winner shortly after, with Canada able to hold on from there and quell England's historic run.
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Speaking after the match, England's head coach John Mitchell said he was focused on taking what positives he could out of the loss.
He said: "We're not driven by outcome. We're disappointed by the result but there was some very good things in our performance tonight. So we'll stick to our processes.
"I guess (the loss) frees us up and allows us all to find more.
"I know this group has got more in them but sometimes it requires the context that we've just received to dig deeper and find something more.
"Maybe everyone in the group can probably review their 'why's and re-establish those as well because I'm sure they're changing and evolving as they experience what we're going through."
The opener of a two-game Rugby Rematch Tour between the growing rivals drew an electric crowd to BMO Field as Canada returned to home soil for the first time since finishing runner-up to the Red Roses at last year's World Cup.
"I think it's a little bit more than just the score at the end because we won on so many levels today," Canada's Justine Pelletier said.
"Off the field, just the crowd, being home, and there's so much going on around. And I think that win at the end on the pitch was just the cherry on (top).
"It was good to beat England, but I think for a few years now we knew that we could beat them. It was just a matter of time and a matter of putting every detail together."
"The bit we always remember is we're at the top, and when you're at the top, people want to bring you down as quick as they can," England captain Megan Jones said, adding that the team take "huge pride" in their unbeaten run.
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Canada's last win over England came in 2016 at the Women's Rugby Super Series.
Mitchell's side will face Canada again next Saturday in Ottawa before completing the away leg of their Global Series fixtures against the USA in New Jersey on October 30.
WXV Global Series: England's schedule and results
- England 48-15 Australia
- England 26-26 Canada
- England 26-19 New Zealand
- Canada 24-21 England
- October 23 - Canada v England (Ottawa)
- October 30 - USA v England (New Jersey)
Scotland suffer Wallaroo walloping
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Scotland Women fell to an eighth consecutive defeat as a rampant Australia ran in five tries in a dominant first half, paving the way for a 56-12 victory in Sydney.
Desiree Miller scored 42 consecutive points, and with her opening try she became the first Wallaroo to have scored 20 Test tries.
The teams will play again in Canberra on Friday.