Alistair Darling described a TV advert based on events this year such as the Year of Homecoming, the Commonwealth Games and the Ryder Cup as showing the tourism agency had been "suborned" by the Scottish Government.
The advert, produced for both domestic and international airing to highlight this year's series of events, concludes with Sir Chris Hoy saying: "So you too can say, 'yes, I was there'."
Sir Chris and his Olympic achievements featured in the recent "lovebomb" speech by David Cameron.
The leader of Better Together accused the SNP of being "ruthless" about exploiting the bureaucracy of the Scottish Government to advance the Yes cause.
He cited the advert, saying: "This is TV advertising for their campaign and VisitScotland has been so suborned they're going along with it."
A spokesman for VisitScotland said: "There was no communication between VisitScotland and Scottish Government officials or ministers regarding the wording in the advert.
"The wording in the advert was created by the advertising agency Union, who included it in their pitch for the creative development. The wording for the poem is to be taken in the context of the events shown within the advert which will give a visitor a memorable 'brilliant moment' and relates to the sporting and cultural events that are included.
"The only overt messages are those that call on visitors to come to Scotland to be part of Homecoming, the Ryder Cup and Commonwealth Games, bringing economic prosperity to the country and returning again and again because of the great experience they have had. It's impossible to be anything but positive around these amazing events."
He added that the advert had been tested across the UK and had received no negative feedback.
SNP MSP and deputy convener of the Scottish Parliament's economy, energy and tourism committee, Dennis Robertson said: "The No campaign is characterised by spreading misinformation about Scotland and distorting reality. It has reached a new low when its chairman, Alistair Darling, makes scurrilous and utterly untrue allegations about the country's tourism agency.
"Tourism is one of Scotland's biggest industries - supporting 200,000 jobs - and the work of VisitScotland is crucial to its success.
"Mr Darling's attack on VisitScotland could only have the effect of undermining this highly effective and important agency, and therefore he has a duty to withdraw his bogus claims and issue a public apology."
Suborn implies bribery or inducement to commit an unlawful act.
A Better Together spokesman said: "We stand by our comments. If VisitScotland want to bring clarity to this situation, let them publish the detailed breakdown of how much money is being spent on this campaign in Scotland compared to the rest of the world."
A Scottish Government spokesman said: "We had no input into the wording used in the advert."