The twelve leaders face charges including rebellion and sedition and, if convicted, some could face up to 25 years in prison.
The semi-autonomous region of Catalonia held an independence referendum on 1 October 2017 and declared its independence from Spain weeks later.
But Spanish authorities declared the vote illegal, and the national government imposed direct rule.
Nine of the defendants have spent months in pre-trial detention and arrived at the court under guard on Tuesday. The remaining three had been free on bail.
The most high-profile of the Catalan leaders on trial is the former Vice-President Oriol Junqueras. His superior - former President of Catalonia Carles Puigdemont - fled abroad and remains in exile in Belgium.
Others include the former speaker of the Catalan Parliament, Carme Forcadell, and two influential grassroots activists, Jordi Cuixart and Jordi Sànchez.
“We are convinced that the unfounded accusations of rebellion and sedition will be proven false. This trial is a huge political failure” Ska Keller MEP
Among the 12 are two former members of the Greens/EFA group in the European Parliament.
Speaking at a news conference in Strasbourg on Tuesday, the Greens/EFA joint leader Ska Keller told reporters, "We call for a fair and independent trial. We are convinced that the unfounded accusations of rebellion and sedition will be proven false.”
“This trial is a huge political failure. We call once more for the end of the provisional imprisonment of the politicians and leaders during the months of the trial.”
Keller went on, "We support the efforts of both the Catalan and Spanish Government to try to establish a sincere and open dialogue in order to find a political solution for Catalonia. A political solution remains the only possible solution for the conflict."
However, speaking at a separate briefing, Spanish centre-right MEP Esteban González Pons, a deputy leader of the EPP group in Parliament, made a robust defence of the rights of the Spanish Government and courts in the affair.
He said, “I say let the courts do their job. There was an attempt [in the 2017 referendum] to split the country and break it into two parts and the Government has acted according to the law.”
“The judges will decide and that is how it should be. They have the ultimate say on whether someone is guilty or not. We must accept what they say and will accept their verdict.”
“This was an illegal referendum. It was a clear rebellion and a violation of constitutional law. Those involved were trying to break up Spain” Esteban González Pons MEP
“It is not up to one part of Spain to decide what should happen to another part of Spain. This was an illegal referendum. It was a clear rebellion and a violation of constitutional law. Those involved were trying to break up Spain,” he added.
The trial begins almost a year and a half after the failed independence bid - which remains controversial.
In Madrid on Sunday, thousands gathered in a pro-unity demonstration against Catalan independence.