Around 200,000 people took to the streets of Barcelona for a candle-lit protest against the detention of two separatist leaders.
Similar demonstrations were held in Girona and other Catalan cities.
It follows the National Court's decision this week to keep Jordi Cuixart and Jordi Sanchez behind bars pending investigations into sedition charges.
The Spanish government has demanded that Catalonia renounce a symbolic declaration of independence, setting it on a political collision course with Madrid later this week.
The government has threatened to put Catalonia, which accounts for a fifth of Spain's economy, under direct central rule if its regional government does not abandon independence by Thursday.
Catalonia's government has rejected Prime Minister Mariano Rajoy's deadline.
"Giving in forms no part of this government's scenarios,"Catalan government spokesman Jordi Turull said.
"On Thursday, we won't give anything different than what we gave on Monday."
The Catalan government accused Madrid of taking "political prisoners" and Catalan leader Carles Puigdemont, in a tweet following the detentions, said: "Sadly, we have political prisoners again."
The phrase was an allusion to the military dictatorship under Francisco Franco, when Catalan culture and language were systematically suppressed.
It carries an emotional resonance given fascism is still a living memory for many Spaniards.
Justice Minister Rafael Catala hit back, saying the jailing of the leaders of the separatist groups, the Catalan National Assembly and Omnium, was a judicial, not a political, decision.
"We can talk of politicians in prison but not political prisoners," he said.
"These are not political prisoners because yesterday's prison ruling was due to a (suspected) crime."
The crisis has deepened divisions at the heart of Spain's young democracy, underlining the complex sense of nationhood in the euro zone's fourth largest economy.
