The pre-dawn operation comes amid simmering tensions between Spain's central government and Catalonia over the wealthy region's independence drive, with separatists saying it amounted to "plundering".
Police cordoned off the museum as workers arrived to remove the disputed art works, including three 15th century wooden caskets.
Several hundred people gathered outside the museum chanted "Hands up! This is a robbery", images broadcast on Spanish television showed.
Some demonstrators briefly scuffled with police as officers tried to move them on.
Catalonia's regional government bought the art in 1983 from the nuns of the Sijena monastery in neighbouring Aragon.
But the regional government of Aragon has since fought to recover the works, arguing they were unlawfully sold, and a court had given the Catalan museum until Monday to hand them over.
Spanish Prime Minister Mariano Rajoy's conservative government dismissed Catalonia's government and suspended the region's autonomy after the Catalan parliament on October 27 declared independence.
"It is best to respect court decisions," Rajoy said when asked about the operation to remove the art.
But Catalonia's ousted leader, Carles Puigdemont, took to Twitter to accuse the Spanish government of "taking advantage of a coup d'etat to plunder Catalonia with total impunity."
Amb nocturnitat i utilitzant una policia militaritzada, com sempre, tot aprofitant un cop d'estat per espoliar Catalunya amb absoluta impunitat. Aquest és el model de país que defensen Ciudadanos, PSC i PP. https://t.co/z7gUPr6yy2— Carles Puigdemont 🎗 (@KRLS) December 11, 2017
Puigdemont, who fled to Belgium after the Catalan parliament declared independence, is running for office in an early election in Catalonia on December 21st which was called by Spain's central government.
He is wanted in Spain for the crimes of sedition, rebellion and misuse of public funds over his government's independence drive.