The Prime Minister was challenged in the Commons by a Labour MP to clarify his future in the event of a 'leave' decision in the in-or-out EU referendum on June 23.
At Prime Minister's Questions, Labour MP Richard Burgon asked: "If the British people vote to leave the European Union, will the Prime Minister resign—yes or no?"
Mr Cameron replied simply: "No."
His response follows widespread speculation that his position as Prime Minister would be untenable if he lost his attempt to keep the UK in the EU.
Boris accuses Government of 'Project Fear' over Brexit
Some Tory MPs have privately suggested that an "out" vote would be a devastating humiliation for Mr Cameron that would force him out of Number 10.
However others have argued that he should remain in office in the immediate aftermath of an exit vote to ensure stability and begin the process of negotiating Britain's withdrawal from the 28-nation bloc.
Senior Tory aides are thought to be desperate to ensure that the historic poll does not turn into a referendum on Mr Cameron's leadership.
He also used PMQs yesterday as an opportunity to make further claims about EU membership.
In response to another question from Labour, the Prime Minister claimed EU membership helped UK citizens benefit from "cheaper air travel, ease of travel and not having any tariffs".
He said: "These are things we take for granted now, but they were simply not the case 40 years ago. "I agree that that is a strong part of the very positive case we should make for remaining in the EU."