Boris Johnson said Britain will remain 'rock-like' in its support for Gibraltar
Often referred to as "The Rock", Gibraltar is a British overseas territory on the southern tip of Spain.
Madrid claims sovereignty over the territory - which it ceded to Britain in 1713 - and the issue has often caused diplomatic tensions with London.
Gibraltar's status after Britain leaves the EU is set to be one of the most contentious issues in complex exit talks expected to last years.
Johnson made his comment about Gibraltar late on Friday after speaking on the phone to Fabian Picardo, Gibraltar's chief minister.
"As ever, the UK remains implacable and rock-like in our support for Gibraltar," he wrote on Twitter.
Picardo has accused Spain of a "disgraceful" attempt to manipulate the Brexit process to further its sovereignty claim over Gibraltar.
According to the EU's draft joint position on exit talks with Britain, no agreement between the UK and the EU may apply to Gibraltar without a separate agreement between Spain and the Britain. The Spanish government said it was satisfied with the position.
In her 2,200-word letter triggering Article 50, Theresa May did not mention Gibraltar once. The BBC reported that Lord Boswell, chairman of the House of Lords EU Committee, said the government must not think of Gibraltar is an "afterthought."
Christian Hernandez, president of the Gibraltar Chamber of Commerce, told BBC Radio Four's Today programme the British government should stand up against "Spanish bullying."
"We don't want to be independent from the UK," he said. "We've made it very clear in the last 100 years, in the last 20 years, in the last 15 years, we want a constitutional relationship with the UK, where we continue to be part of the UK and independence is not something we aspire to."
(Reporting by Estelle Shirbon Editing by Jeremy Gaunt)