A Spanish navy ship tried to divert two commercial vessels heading to the port of Gibraltar by wrongly claiming they were in Spanish waters, Europe Minister David Lidington said.
It is the fifth time since 2011 that Ambassador Federico Trillo has been hauled in over the long-running dispute over the tiny British peninsula off the southern coast of Spain.
Prime Minister David Cameron also raised the incident with his Spanish counterpart Mariano Rajoy on the sidelines of a European leaders' summit in Brussels on Wednesday.
"I am extremely concerned by the actions of a Spanish Navy vessel which sought to redirect two commercial vessels heading to and from the Port of Gibraltar, wrongly claiming they were in Spanish waters," Lidington said.
"The vessels were in international waters off Gibraltar, and the actions of the Spanish Navy vessel constitute a breach of the UN Convention on the Law of the Sea."
He said it was a "cynical attempt by the Spanish government to disrupt Gibraltar's economy, in contravention of international law".
There was no immediate reaction from Madrid.
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.