The European Parliament stripped Carles Puigdemont, the former president of Spain’s Catalonia region, and two other former officials of immunity from prosecution on Tuesday.
The decision could pave the way for the extradition of Mr Puigdemont from Belgium to face sedition charges.
Mr Puigdemont, former Catalan health minister Toni Comin and Clara Ponsati, the region's former education minister, fled Spain in 2017 after they organised an independence referendum that Spanish authorities said was illegal.
Spain charged the three MEPs with sedition and issued a European arrest warrant, but so far has failed to convince Belgium to extradite Mr Puigdemont.
As MEPs, the three former Catalan officials were afforded protection as members of the EU assembly.
Spain's Foreign Minister Arancha Gonzalez Laya said the decision was a sign of "respect for the work of the Spanish justice system".
She said a European politician cannot use their condition to "protect himself from appearing in a national court."
"Problems in Catalonia are solved in Spain, not in Europe," Ms Laya said.
In the vote on Tuesday, 400 MEPs voted to remove Mr Puigdemont’s immunity, 248 were against and 45 abstained.
His legal team have made it known they will appeal, a process that could take years.
"We will not give up," his party, Together for Catalonia, said after the vote.
"The political conflict between Catalonia and Spain has stopped being an internal affair. We have brought it to the heart of Europe to continue denouncing the repression and political persecution of the Spanish state," it said.
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Pro-independence Catalan protesters burn barricades after a previous protest called by the local Republic Defence Committees (CDR) in Barcelona on October 17, 2019. After years of peaceful separatist demonstrations, violence finally exploded on the Catalan streets this week, led by activists frustrated by the political paralysis and infuriated by the Supreme Court's conviction of nine of its leaders over a failed independence bid. / AFP / Pau Barrena -
Catalan protesters gesture behind barricades in flames after a demonstration called by the local Republic Defence Committees (CDR) in Barcelona on October 17, 2019. AFP / LUIS GENE -
An Estelada (Catalan separatist flags) hangs from an overpass as demonstrators march during Catalonia's general strike in El Masnou, Spain, October 18, 2019. REUTERS/Albert Gea -
Catalan demonstrators wearing and waving Esteladas (Catalan separatist flags) chant slogans as they march during Catalonia's general strike in El Masnou, Spain, October 18, 2019. REUTERS/Albert Gea -
Catalan demonstrators chant slogans in front of La Sagrada Familia basilica during Catalonia's general strike in Barcelona, Spain, October 18, 2019. REUTERS/Albert Gea -
An elderly man joins a pro-independence gathering outside the Sagrada Família basilica i on the fifth day of protests over the conviction of a dozen Catalan independence leaders in Barcelona, Spain, Friday, Oct. 18, 2019. AP Photo/Bernat Armangue -
Pro-independence protesters march along a highway in San Vicenc dels Horts, on October 18, 2019, on the day that separatists have called a general strike and a mass rally. AFP / Pau Barrena -
Protesters ride on tractors as they enter the city on the fifth day of protests over the conviction of a dozen Catalan independence leaders in Barcelona, Spain, Friday, October 18, 2019. AP Photo/Manu Fernandez -
Catalan demonstrators wave Esteladas (Catalan separatist flags) as they block roads during Catalonia's general strike in Sant Just Desvern, Spain, October 18, 2019. REUTERS/Juan Medina
In 2017, Catalans overwhelmingly voted in favour of independence, but the government in Madrid declared the vote unconstitutional.
Hundreds of people were injured in a police crackdown on the day of the poll.
Independence leaders were convicted in Spain of the same charge of sedition in 2019 and sentenced to up to 13 years in prison.
