Ian Taylor, chief executive of oil trader Vitol and a regular Tory donor, was subjected to intense criticism when the No campaign revealed in April he had given it £500,000.
His company's track record, which included fines and controversial deals in Iran, Iraq, Serbia and Libya, was pored over by the SNP and other supporters of the Yes campaign, who demanded Better Together return his cheque.
Speaking in the Commons, SNP Westminster leader Angus Robertson even linked Vitol to "murder, wilful killing, rape, and other inhumane acts", because it paid a reported $1 million to the notorious Serb warlord Arkan 20 years ago.
It later emerged that SNP ministers, including Alex Salmond and Nicola Sturgeon, had backed charities partly funded by Taylor.
Six months on, with both sides in the campaign facing criticism for delaying their latest donor information, the Sunday Herald understands Taylor has told Better Together his donation was a "one-off".
London-based Taylor and his wife Cristina have given almost £700,000 to the Tory Party since 2006.
The loss of Taylor as a potential regular donor will be a blow to the pro-union side in the independence referendum debate.
His £500,000 accounted for almost half the £1.1m received by Better Together up to April.
Yes Scotland received £1.7m.
Both Better Together and Yes Scotland are this weekend under fire for delaying the release of their latest donor information, despite claiming to be committed to transparency.
The two sides had been expected to volunteer their income and the names of those giving more than £7500 this week, exactly six months on from their first declarations.
However, it is understood Better Together intends to postpone its figures until after the SNP Government publishes its White Paper on independence next month.
Yes Scotland is refusing to release its figures until Better Together releases its details.
The foot-dragging was last night condemned by Sir Alistair Graham, former chairman of the Committee on Standards in Public Life.
Neither side is under any legal obligation to reveal its donors until 16 weeks before the ballot.
Yes Scotland's delay is in spite of a pledge given last November by its then director of communications, Susan Stewart, who told the Sunday Herald donations would be published "every six months".
Blair McDougall and Blair Jenkins, respective chief executives of Better Together and Yes Scotland, also told Westminster's Scottish Affairs Select Committee in 2012 that they were committed to openness on the issue.
Jenkins told MPs: "We will certainly be making clear where our funding has come from and publishing all the details of that."
McDougall expected to declare donations "on a voluntary basis".
Sir Alistair said the delays in disclosure by both sides were "most unfortunate". He said: "If they said they will apply certain rules to themselves they should continue that self-discipline, otherwise people will fear the worst about where the money is coming from."
Better Together said: "We are committed to voluntarily releasing the names of people who have made contributions. We will make another disclosure in due course."
A Yes Scotland spokesman said Jenkins had written to Better Together chair Alistair Darling in January suggesting simultaneous publication of the campaigns' donors, but had received no response.