Boris Johnson has pledged that Gibraltar will not be sacrificed in Brexit negotiations and rejected claims that it could become a "bargaining chip".
The Foreign Secretary hit out after Labour's Sir Keir Starmer urged ministers to block attempts by the EU to give Spain a veto on Gibraltar's future.
"Gibraltar is not for sale," Mr Johnson told The Sunday Telegraph. "Gibraltar cannot be traded. Gibraltar will not be bargained away."
He added: "The policy of the Government remains fixed and firm. The sovereignty of Gibraltar cannot be changed without the express consent of the UK and the people of Gibraltar."
Praising Gibraltar's "vibrant business centre", Mr Johnson said its harbour remained a "key NATO asset" because it can take nuclear submarines.
"The UK Government can be counted on to stick up for those interests - for instance in insurance and maritime services - which create jobs not just in Gibraltar but in the wider region of southern Spain," he said.
"The status of Gibraltar has been unchanged since 1713. It made no difference when the UK joined the Common Market in 1973 and when Spain was not yet a member. It should make no difference today.
"So let us go into these discussions with goodwill and optimism and get a deal that is good for the UK, good for Spain, and good for the people of Gibraltar."
Spain has a long-standing territorial claim on Gibraltar, which has been held by the UK since 1713 and has the status of a British overseas territory.
Gibraltar's chief minister Fabian Picardo, also speaking to The Sunday Telegraph, hit out at the threat of a Spanish veto on the Rock's future.
"It is a slap in the face for the people of Gibraltar who are the ones who most vehemently supported remaining in the European Union," he said.
"It is unfair, it demonstrates that Spain will use any opportunity to try and advance its claim to Gibraltar."
The heat is being turned up on the Government after criticism of Theresa May for failing to mention Gibraltar in her Article 50 letter to European Council president Donald Tusk.
Downing Street has said the Prime Minister made clear in her Commons statement on Article 50 on Wednesday that the Government was "absolutely steadfast in our support of Gibraltar, its people and its economy".
But former Tory MP Lord Boswell, chairman of the House of Lords EU Committee, has said it was "unfortunate that the Prime Minister's letter to Donald Tusk, triggering Article 50, made no mention of Gibraltar".
He said that "in the absence of any clear commitment to defend Gibraltar's interests by the Prime Minister, the door has been opened for the EU to present it as a disputed territory".
At the same time, negotiation guidelines set out by Mr Tusk appear to put the future of Gibraltar at stake by giving Spain a veto in discussions about the Rock's future.
"After the UK leaves the Union, no agreement between the EU and the United Kingdom may apply to the territory of Gibraltar without the agreement between the Kingdom of Spain and the United Kingdom," he wrote.
Pro-Gibraltar Tory MP Andrew Rosindell told Sky News the Government must stand firm against the Spanish threat.
"Any Brexit agreement without including Gibraltar means there can be no agreement," he said.
"British people must stand together. We cannot be bullied by Spain.
Any deal must apply equally to the whole British family and that includes Gibraltar. There can be no compromise on this."