The new First Minister was dealt a hammer blow as Michael Shanks snatched the Rutherglen and Hamilton West seat with 17,845 votes.
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And the margin of victory in the first major electoral test for Mr Yousaf is likely to spark fears of a General Election collapse for the Nats.
Sir Keir Starmer’s Labour grabbed the seat won by ex SNP MP Margaret Ferrier in 2019, before she was ousted following her Covid lawbreaking.
Labour’s Michael Shanks won 17,845 votes or 58.4 per cent of the vote share and SNP’s Katy Loudon tallied up 8399 votes.
The results put Labour 31 points ahead of the SNP, who won the seat in 2019 with a share of 27.9 per cent.
Tory Thomas Kerr was in third with 1192 votes and failed to win back their deposit with just 3.9 per cent of the share.
Following the result, Shanks said: “It is the honour of my life to have been elected as the MP for Rutherglen & Hamilton West.
“Thank you to all those who helped deliver this result.
“The largest thanks goes to all those who have put their trust in me.
“My promise in return is a simple one: I will be your champion.”
He added: “This isn’t as good as it gets and under the leadership of Anas Sarwar, people are once again listening to the Scottish Labour Party and know from tonight that change is possible.
“Across the UK, Sir Kier Starmer is showing a relentless focus on delivering for working people and offers a clear alternative to the morally bankrupt Tory party.
“We’ve had more than enough of managed decline, more than enough of division and distracted, chaotic government.
“It’s time for change. That change can’t come fast enough.
“Labour can kick the Tories out of Downing Street and deliver the change that people want.”
Sir Keir Starmer, leader of the Labour Party, reacting to a Labour gain from the SNP in the Rutherglen and Hamilton West by-election, said: “This is a seismic result.
“People in Rutherglen and Hamilton West have sent a clear message – it is time for change. And it is clear they believe that this changed Labour Party can deliver it.
“I have always said that winning back the trust of people in Scotland is essential.
“Tonight’s victory is the culmination of three and a half years of hard work in years of hard work and humility on that journey.
“I am grateful to everyone who has put their faith in us today – we will work every day to repay it.
“Voters across Scotland and across Britain want a government determined to deliver for working people, with a proper plan to rebuild our country.
“They want to move on from two SNP and Tory governments that offer only more division, more chaos and more infighting.
“The country deserves a government firmly on their side and focused on their priorities – and Labour will deliver that for them.”
Prof Sir John Curtice called the outcome a “remarkably good result” for Labour and said “if this sort of swing was replicated north of the border Labour would become the dominant party” after the next General Election.
There was a total turnout of 37.19 per cent with a total number of 30,531 votes cast from an electorate of 82,104.
This is well below the 66.7 per cent at the 2019 election, but by-elections tend to have lower turnouts.
The 2021 Airdrie and Shotts by-election which saw voters elect SNP MP Anum Qaisar had a turnout of 34.3 per cent.
The loss comes as a major blow to Mr Yousaf, who earlier in the day had been celebrated on the front of the New York-based Time as one of ten “trailblazers shaping the future” across the world.
But back in Scotland, SNP chiefs crashed back down to earth with the long-awaited by-election.
Fromer MP Margaret Ferrier was booted out after travelling to London from Glasgow while knowingly having the Coronavirus.
At the time, mass vaccinations had not started and restrictions in Scotland included the “rule of six”, with indoor household gatherings banned. The MP took a Covid test on September 26, 2020, due to a “tickly throat”.
But while awaiting her results, she went to a church and gave a reading to the congregation, before spending more than two hours in a bar.
She was suspended from the SNP, stood as an Independent and then suspended from the Commons for 30-days.
Her constituents then signed a recall petition forcing her out and triggering a by-election.
This by-election was the first test for First Minister Humza Yousaf who took over the job back in March this year.
In the last few desperate hours of campaigning, Mr Yousaf and Scottish Labour leader Anas Sarwar posed for pictures after dashing west to the key constituency to rally voters following First Minister’s Questions at Holyrood.
The scale of Labour’s win will be pored over in the coming days — with polling experts saying it could signal how many of the 40-plus Nats seats they can grab at next year’s General Election.
As Nats chiefs admitted they had lost, the source pointed to issues including the Ferrier factor, and “general party difficulties” – in an apparent nod to the police’s SNP finance probe and the bombshell arrest of figures including Nicola Sturgeon – meaning it was “a tough one for us”.
The by-election had prompted desperate efforts by the SNP to cling on the seat Ferrier won for them four years ago.
But the party has been hit by a poll slump over the past year and the Rutherglen ballot was widely seen as a head-to-head fight with Labour, who only secured one of Scotland’s 59 Westminster seats in 2019.
Amid the cost of living crisis, Sir Keir’s party tried to focus voters’ minds on electing a change to the SNP and Tories.
Polls closed at 10pm with bookies predicting Labour’s Mr Shanks to win with 1/20 odds to be elected MP, with the SNP’s Ms Loudon rated a 10/1 outsider.
Earlier today the betting firm’s spokesman John Hill said: “Labour are long odds-on to be celebrating.
“Their price shortened over the past 24 hours from 1/16 to 1/20.
“The SNP are tipped to be second but it would be a big shock if Labour were not to win this important by-election.”
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