Main highways, including Spain's export route to France and the rest of Europe, were cut in about 60 places causing widespread disruption in the region which has been plunged into uncertainty over its now-deposed government's bid to split from Spain.
A few hundred protesters managed to block the tracks at Barcelona's main Sants station.
Authorities said high-speed train links with France were disrupted, with four out of eight daily trains affected.
Today, Spain's Constitutional Court struck down the declaration of independence made by Catalan lawmakers on October 27 -- which led Madrid to dismiss the regional government and assume direct control of Catalonia -- declaring the secession bid "unconstitutional and void."
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Madrid is organising new regional elections in Catalonia for next month as it tries to stem the fallout from Spain's deepest political crisis in decades.
Juan Jose Gil of the National Transport Federation said that the AR-7 highway -- "the main transport route for Spanish exports" -- was cut in both directions.
The route is used by an average of 9,000 heavy goods vehicles each day, he said.
French authorities said that the border was blocked at the Spanish town of Puigcerda and that other major routes were cut around Figueres, north of Barcelona
Juan Antonio Puigserver, an interior ministry official, said participation in today's strike had been "minimal" in most sectors.
A judge in Madrid last week ordered eight separatist politicians to be remanded in custody over their secession drive.
Deposed Catalan leader Carles Puigdemont, who fled to Belgium where he is facing extradition back to Spain, yesterday criticised the EU for backing Spanish Prime Minister Mariano Rajoy in the crisis.
Belgian Prime Minister Charles Michel today denied that his government was "in crisis" over Puigdemont's presence, after Flemish separatist members of his coalition government spoke out in support of Catalan independence.
More than 2,000 businesses have moved their legal headquarters out of Catalonia as the turmoil drags on.
Today's walkout was called by the pro-independence CSC union but lacked support from Spain's two largest unions.
Waving pro-independence banners and Catalan flags, demonstrators called for the release of sacked government officials and separatist lobbyists.
Isabel Nistal, 33, said she and her husband had showed up "to ask for the freedom of our government, half of which is in prison and the other half in exile."
Commuter Edison Hincapie, 50, was unconvinced.
"I understand their motivation but ultimately this affects Madrid little, it affects those of us living here."
Fresh elections will be held in Catalonia on December 21 and Rajoy called today for "massive participation" in the vote.
Puigdemont has called for a united separatist front to participate, but a former government ally, the leftwing ERC party, yesterday ruled out running on the same ticket.
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