Buckingham Palace has written to the press watchdog to register a complaint over claims that the Queen voiced strong Eurosceptic views during a lunch with the former deputy prime minister.
The Sun said she vented her anger with Brussels at the pro-EU politician Nick Clegg during a lunch at Windsor Castle in 2011.
The front page story was written by The Sun's political editor Tom Newton Dunn.
The story describes the encounter between the Queen and Clegg, which was also attended by a "hanful of other ministers".
The Queen is said to have told pro-European Clegg, then the deputy MP, that she believed the EU was heading in the wrong direction.
The source of the story, described as a "senior political source", said: "People were left in no doubt about her views on Europe".
A Buckingham Palace spokesman said: "We can confirm that we have this morning written to the chairman of the Independent Press Standards Organisation to register a complaint about the front page story in today's Sun newspaper. The complaint relates to Clause 1 of the Editors' Code of Practice."
Clause 1 of the Editors' Code concerns accuracy. If the story does prove to be untrue The Sun could be forced to publish a critical adjudication on its front page.
The Sun said: "The Sun stands by its story which was based upon two impeccable sources and presented in a robust, accessible fashion. The Sun will defend this complaint vigorously."
It is thought to be the first time that the Queen has complained to IPSO, who said it had received another complaint about the story.
The Duke of York has complained to IPSO previously about a story in the Daily Mail after the newspaper sent a helicopter over the grounds of his home in June 2015 to take photographs of daughter Princess Eugenie's 25th birthday preperations.
The Daily Mail was ordered to take remedial action by IPSO after breaching Prince Andrew's privacy
The IPSO complaints handling team will first seek evidence from The Sun and The Queen and seek to broker a resolution to the issue. If no agreement can be reached between the pair the matter will go to the IPSO complaints committee to judge whether there has been a breach of the code.
Under IPSO rules complainants are normally asked to approach the newspaper directly in the first instance.
Queen backs Brexit – p1 tomorrow pic.twitter.com/ioIfa9GpbK
— Tony Gallagher (@tonygallagher) March 8, 2016