Len McCluskey, the general secretary of Unite and one of Jeremy Corbyn’s closest allies, said the election of the Labour leader had changed his view.
Mr McCluskey also said he believed Labour would win the next general election, but only with a minority in parliament - opening the door to negotiations with the SNP to form a government.
The man dubbed ‘Red Len’ said Unite had taken a "neutral position" in the 2014 referendum campaign because its membership was "split down the middle" on the issue.
But he said Scotland had been "subjected to rule by Westminster" by "neo-liberal" Labour and Conservative-led government, added: "It is why Labour lost so many of their votes to the SNP, the SNP were seen as a more radical social democratic party.
"If I had been in Scotland at the time I would have thought 'You know what, I want to just cut away from London and Westminster'.
"I wouldn't have seen the light at the end of the tunnel. Now we have Jeremy Corbyn and a Labour government that can give a truly transformative policy to all of our nations, Wales, Scotland, and England.
"Therefore if there was an independence referendum tomorrow I would vote No."
Mr McCluskey criticised then-Labour leader Ed Miliband for joining the Better Together campaign with the Tories, and forgot Scottish Labour leader Jim Murphy’s name as he criticised the party’s campaign in the disastrous 2015 general election, that produced an SNP landslide.
After Mr Murphy’s name was shouted out by an audience members, Mr McCluskey added: "Fancy me forgetting Jim.
"Jim Murphy, who was at the epicentre of New Labour, was put in charge of Scottish Labour to win back the Scottish working class, it was doomed to failure."