The First Minister was accused of "gross incompetence" over the sale of land bought in preparation for the doomed Glasgow Airport Rail Link (GARL).
The plots were sold back to their orginal owner, car park operator Airlink Group, at a loss of £790,000 to the taxpayer after the GARL project was scrapped.
At Holyrood yesterday, Scots Labour leader Johann Lamont challenged the First Minister to explain the deal - revealed in The Herald last week - and asked if there was a "connection" with Airlink boss John McGlynn's backing for a Yes vote.
Her comments provoked a furious reaction, with Mr Salmond accusing her of "smearing" the businessman and Mr McGlynn calling for an apology.
The land earmarked for GARL was bought for £840,000 in 2008, then sold back to Airlink for £50,000 this year after it failed to find a buyer at auction.
The sale ended any realistic prospect of reviving the rail link and made Mr McGlynn a profit of £790,000.
Speaking at First Minister's Questions, Ms Lamont said the businessman, a former Conservative donor had "been on something of a political journey" adding: "Now he supports the Yes campaign.
"Since then he's been appointed to the Scottish government national economic forum.
"And since then, he has bought back the land from the Scottish government for just £50,000 and made a profit of £790,000."
She asked: "Is there some connection here, or has Mr McGlynn just benefited from the first minister's gross incompetence with public funds?"
Mr Salmond stressed ministers were not involved with property deals, adding: "What was that about if it was not a fairly blatant attempt to smear a perfectly respectable Scottish businessperson?"
Mr McGlynn, whose new Airlink offices in Glasgow were opened by the First Minister last year, demanded an apology.
He said: "I find it preposterous that anyone should link my personal expressions of sympathy for fiscal autonomy with my commercial activities.
"For the record, I have never expressed support for Yes Scotland or the Yes campaign despite statements made by Johann Lamont.
"I would be delighted to meet her to discuss the facts in full after which I hope that she might find herself in a position to make an apology to me."
He said the deal was "above board, fully transparent, in the public domain and open to scrutiny".
Last year Mr McGlynn said he would "most likely" back independence.
A Government spokesman said: "As has been public knowledge for several years, the Scottish Government is not permitted to hold on to surplus land as a means of speculating on land values. Consistent with Treasury rules we were obliged to dispose of this site.
"The land was eventually sold following a public auction, which was open to all bidders."