Ambassador Federico Trillo was hauled in to explain why a Spanish state research vessel and its police escort entered the waters off the tiny British peninsula on Tuesday, the Foreign Office said in a statement.
It comes barely four months after Trillo was summoned to discuss a day-long stand-off also involving a Spanish research vessel, which defied repeated Royal Navy orders to leave Gibraltarian waters.
"I am extremely concerned to hear of another incursion into British Gibraltar territorial waters on 1 April by a Spanish state research vessel, which sought to undertake survey activity," said Britain's Europe minister, David Lidington.
"Not only were the actions of the survey vessel unlawful, but it was accompanied by a Spanish Guardia Civil vessel whose dangerous manoeuvring presented a significant safety concern on the waters."
He added: "I strongly condemn this provocative incursion and urge the Spanish government to ensure that it is not repeated."
It is the fourth time that Britain has publicly summoned Spain's ambassador over Gibraltar since Prime Minister Mariano Rajoy took office in December 2011.
Spain ceded Gibraltar to Britain in perpetuity in 1713 but has long argued that it should be returned to Spanish sovereignty, and the territory remains a source of diplomatic tensions.
Relations between London and Madrid became particularly strained last year after Gibraltar dropped 70 concrete blocks into the sea in July, in what its government said was an attempt to create an artificial reef.
The move had the effect of also blocking Spanish fishing boats from operating close to the airport runway, and Madrid responded by introducing stringent border checks.
On November 19, the Foreign Office summoned the ambassador to explain an incursion by the oceanographic survey ship Ramon Margalef, which it said came within 250 metres (yards) of the entrance to Gibraltar harbour.
Barely a week later, Britain made a formal protest to Spain after its officials opened British diplomatic bags at the border with Gibraltar. Prime Minister David Cameron accepted Madrid's explanation that a junior official was to blame.