"I am with the people, with the rebels and against Gaddafi's regime," Abdulhafed Gaddur, who has been a Libyan diplomat in Italy since 1990, told the Corriere della Sera daily in an interview.
"I will stay in my post until a new Libya and its new government make their choices," said Gaddur.
Italy was Libya's top trade partner before the start of the conflict there.
The ambassador signed a document in February drafted by defectors from the regime pledging support for the Libyan people but had not spelled out his exact position and his status is unclear even though he retains his title.
An unsourced report in business daily Il Sole 24 Ore after international talks on Libya in Rome on Thursday said that the anti-Gaddafi leadership had asked Italy to replace the ambassador with their own nominee.
Italy is one of four countries along with France, Gambia and Qatar that has recognised the rebel's National Transitional Council.
US Secretary of State Hillary Clinton this week called on all countries to deepen Gaddafi's isolation by expelling all diplomats from the regime.
Britain announced the expulsion of the Libyan ambassador on London earlier this month, following attacks on the British embassy building in Tripoli.