The protesters have decried the trial of 12 Catalan separatists, saying "self-determination is not a crime." Prosecutors have accused the separatists of "rising up in a violent and public manner," allegations they deny.
The march was led by a line of protesters holding a long banner that read, "Self-determination is not a crime." Other banners described the Catalan leaders on trial as "political prisoners."
"It's a political trial, full of manipulations to sentence them for something that isn't a crime," said one protester, who leads a group of "castellers," or human towers. "They just want to lock them up and as they know that voting isn't a crime, they're inventing violence that never happened."
Earlier this week, Spain's Supreme Court opened the trial, with nine of the defendants having been held in detention without bail since late 2017.
The defendants are on trial for their involvement in a botched independence bid. The charges include misuse of public funds, sedition and rebellion, the last being the most controversial given it is legally defined as "rising up in a violent and public manner."
The defendants include former Catalan Vice President Oriol Junqueras and former regional parliament speaker Carme Forcadell, who read the legislature's short-lived declaration of independence.
But they have denied using violence. "If you read, listen and observe our actions, no one could have the least doubt that we reject violence," Junqueras said at his hearing earlier this week.
Despite Saturday's protest, many in Spain back the trial. Earlier this month, Madrid saw mass right-wing protests against Prime Minister Pedro Sanchez, of the Socialist Party, over his negotiations with Catalan separatists.