Boris Johnson, the Foreign Secretary, said on Monday the territory's sovereignty "cannot conceivably change" unless consent is given by both Gibraltar and Britain.
Howard likened May to former Prime Minister Margaret Thatcher, who sent troops to the Falkland Islands in 1982 after Argentina invaded the South Atlantic British territory.
The incident comes after Spain's Foreign Minister Alfonso Dastis told the United Kingdom to calm down after the former Conservative leader Lord Howard suggested Britain could go to war over the Rock.
The PM has reiterated her message of support in a phone call to Gibraltar's chief minister Fabian Picardo, who declared Gibraltar's 32,000 citizens would not be treated as "bargaining chips" in Brexit negotiations. Some 10 thousand persons go to work daily in Gibraltar, that depends on its small land border with Spain to trade and receive visitors and workers.
Sir, - Perhaps the draft European Union guidelines for the Brexit negotiations should include a clause relating to Northern Ireland equivalent to that applicable to Gibraltar?
Mrs. May said they remain absolutely dedicated to working with Gibraltar for the best possible outcome on Brexit, and will continue to involve them fully in the process.
Howard's comments followed an interview on Sunday by Michael Fallon, the UK's defence secretary, in which he said: "Gibraltar is going to be protected all the way because the sovereignty of Gibraltar can not be changed without the agreement of the people of Gibraltar and they have made it very clear they do not want to live under Spanish rule".
A Downing Street spokesman said Monday: "All that Lord Howard was trying to establish is the resolve that we will have to protect the rights of Gibraltar and its sovereignty".
The Prime Minister spoke to Gibraltar's leader Fabian Picardo to tell him the United Kingdom remained "absolutely dedicated to working with Gibraltar for the best possible outcome on Brexit". It said that one of the two patrol vessels that Britain's Royal Navy keeps in Gibraltar chased the Spanish ship off.
As a result, Madrid has always been seen as an obstacle to an independent Scotland joining the European Union after Brexit.
Lord Howard's comments were raised in the House of Lords, where the foreign minister was called on to "distance herself" from the idea that the United Kingdom could go to war with Spain.