The comments were made on Saturday after it emerged that British officials had flown to Madrid to discuss the issue.
A Cabinet Office official working on Scottish constitutional issues and Andrew Dunlop, Downing Street's Scotland adviser, were apparently invited to visit by the Spanish government.
Details of the trip were met with fury by Alex Salmond who lambasted the meeting as an attempt to sabotage the ‘pro-independence campaign, which he leads.
He told Scottish daily the Sunday Herald: "David Cameron's Spanish stitch-up exposes the fact that anything the Prime Minister of Spain says about Scotland is at the behest of the Westminster Tory government.
"We now know that the Prime Minister sanctioned Downing Street plotting with their Spanish counterparts to interfere in Scotland's referendum, and they are clearly working hand in glove."
The trip follows damaging comments by Spanish Prime Minister Mariano Rajoy about Scotland’s EU membership in the case of a vote for national independence.
In what has been widely interpreted as a warning shot across the bow of Catalonia, the prosperous region currently planning a referendum on independence from Spain, he stated that if part of an EU state chose independence, it would put itself outside the EU and need to apply for re-entry.
He added: "This is a fact - it's neither a value judgment nor an opinion, it's simply a fact."
It is a fact contested by Salmond and his Catalan counterparts who claim that membership deals could be negotiated during the independence process, while their countries are still part of the EU.
It is believed that Spain could choose to make membership difficult for Scotland, by using its veto powers to slow the process as a warning to Catalonia.
A Downing Street spokesman told the Sunday Herald: "It is quite common for UK Government officials to meet officials in other European Governments to discuss issues of common interest.“
“Any suggestion that our policy officials can tell the Spanish Government how to run their own domestic affairs is laughable."