Distillers William Grant & Sons, the makers of Glenfiddich malt whisky, are understood to have given more than £100,000 to the pro-Union Better Together movement, as well as cash to other groups backing a No vote.
The 127-year-old company - which last year had a turnover of more than £1 billion - is one of the few big distillers still in family hands. Its global brands include The Balvenie, Tullamore Dew, Hendrick's Gin, Sailor Jerry rum, and Grant's.
The company gave a clue to its thinking last month when its Girvan distillery played host to Better Together's Alistair Darling when he gave a pro-Union speech.
A spokesman for Grant's said the £5bn whisky industry enjoyed "substantial support from the UK Government", including promotion through its embassy network, as well as a lack of trade barriers as a result of European Union membership. Losing these under Scottish independence could "severely damage" the industry, which employs about 35,000 people.
One of Better Together's other big donors, Donald Houston, owner of the Ardnamurchan Estate, who last year gave £600,000 personally and via his firms, also owns a distillery.
The vote of no confidence from such an integral part of the Scottish economy is a blow to Alex Salmond and the Yes campaign, and comes just days after a YouGov poll suggested support for independence was slipping.
Grant's is also headquartered in Dufftown in Moray, the constituency of SNP agriculture secretary Richard Lochhead, who is in charge of promoting Scottish food and drink overseas.
On Tuesday, the Electoral Commission publishes a detailed breakdown of recent giving.
Better Together has so far declared donations of £2.8 million between May 2012 and December last year. Its more recent donations include £1m from the Harry Potter author JK Rowling.
Yes Scotland has so far declared cash donations of £4.5m, of which £3.5m came from Chris and Colin Weir, the Largs couple who won £161m on the EuroMillions Lottery in 2011. A Yes Scotland source indicated it would not be revealing much additional cash on Tuesday.
Both campaign groups are limited to spending £1.5m during the final 16 weeks of the referendum campaign.
However, spending on staff does not count towards this cap, provided people have a direct employment contract with the campaign group.
It is understood Better Together is now hiring staff to help organise events and distribute material.
Although it has not explicitly backed a No vote, trade body the Scotch Whisky Association (SWA), recently called for assurances that the industry would enjoy a "business, regulatory, and export environment at least as supportive" under independence as in the UK.
A spokesman for William Grant & Sons Holdings Limited said: "We can confirm that the company has made these donations.
"We support the stance of the SWA over independence and would refer you to their recent statement that the Scotch whisky industry enjoys substantial support from the UK Government and its worldwide embassy network and from lack of trade barriers from within the EU."
A Better Together spokesman said: "We are delighted to have received this support."