The Spanish Foreign Ministry has summoned Cuba's ambassador to Madrid after a Spanish opposition politician was denied access to the island.
Relatives of jailed dissidents have been holding protests in Havana
MP Jorge Moragas of the Popular Party (PP) was expelled on Friday on his arrival at Havana airport.
He had announced he would show his support "support and solidarity" for Cuban dissidents opposed to President Fidel Castro's regime.
The incident risks halting a thaw in Spanish-Cuban relations.
Spanish Foreign Minister Miguel Angel Moratinos said the expulsion was "unacceptable".
"The Foreign Ministry has urgently called the Cuban ambassador to Spain to ask her for an explanation," a ministry statement said.
"The government reaffirms its wish to continue working on democratisation and the defence of rights and freedoms in Cuba," it added.
The Dutch government, too, has condemned the expulsion of two MPs who travelled to the island on Friday for the same reason and were forced to reboard the same Air France plane bound for Paris.
But Cuban authorities said the visit by the three politicians was a "crude political provocation", and accused Mr Moragas of trying to enter the country "fraudulently and illegally" on the grounds that he was holding a tourist visa.
At a press conference in Madrid, Mr Moragas said: "I was expelled because the position of the European Union with regard to Cuba is being debated and the Spanish government is establishing a unilateral dialogue with Cuba to modify it."
PP Secretary General Angel Acebes said Prime Minister Zapatero was "closer to the dictator than to the democrats who are fighting for freedom".
Last year, the EU imposed diplomatic sanctions on the island after 75 dissidents were jailed.
It also criticised the Cuban government for restoring the death penalty following the execution of three men who hijacked a ferry in an attempt to leave the country.
The Cuban government said the men had committed "very grave acts of terrorism."
The Spanish government of Socialist Prime Minister Zapatero says the EU's policies towards Cuba are ineffective, and is currently trying to restore dialogue with Havana.
Former Spanish Prime Minister Aznar, a close ally of US President George W Bush, had virtually frozen relations with Mr Castro, who had dubbed him "a little Fuehrer".