The crowd gathered in the Catalan capital, shouting slogans calling Spanish police an "occupying force" and urging them to leave the northeastern region.
The protest came as several small labor unions and grassroots pro-independence groups urged workers throughout Catalonia to go on partial or full-day strikes and as the region's leaders ponder a possible declaration of independence, two days after the referendum that turned violent and that Madrid deemed illegal and invalid.
"They brought violence with them. They have beaten people who were holding their hands up. How can we not be outraged?" Demonstrators arrived by foot, walking along empty boulevards and streets closed off by municipal police as tourists watched from distance.
Protesters blocked several roads in the city and bus and taxi services were affected.
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The strike was not backed by Spain's two main unions, the UGT and CCOO groups, and there were no reports of disruptions affecting big industry or Barcelona's airport.
Office worker Antonia Cuello, 37, was in two minds about the industrial action.
"On one side it is a hassle to try to get to work in the midst of a strike," she said. "We are suffering this because a few decided to behave in an improper way. On the other hand, I understand the circumstances surrounding the strike."
Port workers also held a demonstration outside the regional headquarters of Spain's ruling Popular Party, while firefighters planned a rally outside the Interior Ministry's regional office in Barcelona. Protests were also to be staged outside polling stations where police acted with force to try to prevent Sunday's poll being held.
The police action prompted criticism worldwide although the European Union and most governments backed Spain's stance in what is its most serious political crisis in decades.
Nigel Farage, one of the leaders in Britain's vote to leave the EU, condemned the bloc's failure to clearly condemn the police violence.
Cyprus said Spain's national sovereignty and territorial integrity needed to be respected, arguing that the referendum on Catalonia's independence was carried out "in violation" of the Spanish constitution.
Turkey's foreign ministry spokesman Huseyin Muftuoglu also said respect for Spain's territorial integrity was the main "principle," adding that it was important that both sides abide by Spain's laws and avoid violence.