Sitting on benches in the ornate room of Madrid's Supreme Court, the 12 faced a row of judges and a discreet Spanish flag in proceedings broadcast live on television, as their lawyers spoke at the sides.
All are in the dock over an independence referendum held on October 1, 2017 despite a court ban, as well as a short-lived declaration of independence, which sparked Spain's worst political crisis since the death of dictator Francisco Franco.
"This case targets political dissidence," said Andreu Van den Eynde, the lawyer of two defendants including former Catalan vice president Oriol Junqueras, the trial's main protagonist who faces up to 25 years in jail.
He accused authorities of violating fundamental rights in a process that has forced Madrid to defend its judiciary, with Supreme Court President Carlos Lesmes blasting "a big smear campaign."
Before the start of the trial, separatist officials demonstrated near the courthouse where more than 600 journalists are accredited, holding a banner that read: "Deciding is not an offence".
Separatists in Catalonia want to be able to hold a referendum on their future and have dismissed the trial as a politically-motivated "farce".
Pro-independence protesters in the region briefly blocked several roads before dawn, setting fire to tyres and holding up traffic.
Protests have been called in Barcelona, the Catalan capital, at 7 pm (1800 GMT).
But many Spaniards support the proceedings, still dumbstruck over the actions of Catalonia's then regional executive in October 2017 when it tried to break from the country.