GMB Scotland, the third-largest union north of the Border after Unison and Unite, last night confirmed that following a lengthy consultation with its 56,000 members, its formal position would be to support a No vote next September.
The union will now consider how much money to commit to its position - in theory it could spend up to £150,000 to promote a No in the final phase of the campaign.
The GMB decision is a blow to Alex Salmond and the Yes Scotland campaign, who are looking for the support, money and manpower of the unions.
GMB Scotland is the fifth and by far the largest union to commit to a No vote so far, following Aslef, Community, Usdaw and the NUM.
Last week, the Prison Officers' Association in Scotland became the first union to back a Yes vote, prompting Yes Scotland chief executive Blair Jenkins to claim a Yes vote was the "natural choice" for all trade unions. Len McCluskey, the UK general secretary of Unite, also said this week that the arguments for a Yes vote were "seductive", although this may have been calculated to irritate Labour after the party condemned Unite's heavy-handed tactics in the Grangemouth dispute.
Harry Donaldson, general secretary of GMB Scotland, said the union's decision had been reached after a long consultation with Scottish members.
While some were undecided, and some wanted independence, the "overwhelming" opinion was for a No vote and greater devolution.
He said a key consideration had been the potential impact of independence on jobs in defence, shipbuilding and manufacturing.
Feedback from members had informed a final decision by the union's regional council.
"Our view was that we remain within the United Kingdom, however that was not a green light for the status quo," he said.
"For our vision of social and economic change, we want to see a programme for delivering this.
"So we will be campaigning on the basis of much more power to the people, to local authorities and communities."
A spokesman for the Better Together campaign said: "This is now the fifth major trade union to come out in support of a No vote next September.
"It is a clear sign that the bodies who represent workers in Scotland believe that we are stronger when we work together and that the principal of solidarity across the UK is much more powerful an argument than throwing up borders between people where none currently exist. We welcome this decision."
A Yes Scotland spokesman said: "At the heart of this debate is how best we can build a fairer, more socially just Scotland which matches the values, priorities and aspirations of people who live and work here.
"Increasing numbers of people, including trade unionists, are coming to realise that only a Yes vote will deliver all the tools we need to make this possible."