Demonstrators cut the AP7 motorway at La Jonquera near the city of Girona in eastern Spain, a day after a general election.
Around 500 people cut the highway - an important truck route from France to southern Spain - at the border point of La Jonquera, some on the French side and the rest in Spain, a Catalan police spokesman said, Presstv Reported.
Traffic was being diverted to another road and protesters were setting up a stage.
Several demonstrators, some of them with their faces covered, held up banners which read "Everyone to La Jonquera" in Catalan.
Electronic billboards warned the highway was closed due to a protest.
The demonstrators were equipped with cooking utensils, gas cylinders, foldable tables, food, coffee and water.
The protest was called by a new organization called "Democratic Tsunami," which said it would last for three days.
"This mobilization is a cry to the international community so that it makes the Spanish state understand that the only possible path is to sit down and talk," the group said in a message to its followers on encrypted messaging service Telegram.
Radical separatist group CDR also called on its supporters to head to La Jonquera to block the highway.
Anti-riot officers from the Catalan regional police were in a toll area four kilometers away from the Spain-France border protest, the police spokesman said.
Catalonia has been rocked by mass protests since long prison sentences were handed down in mid-October to nine separatist leaders who spearheaded a failed independence bid in 2017.
The day the court issued its verdict, "Democratic Tsunami" sent thousands of people to block access to Barcelona airport in a protest that ended in clashes between demonstrators and police.
The group, which says it favors peaceful civil disobedience, said last week that it would organize a three-day protest starting on Monday, which would be its "most ambitious" action.
The campaign for Catalan independence has been mostly peaceful for years, but some protests turned violent last month, with a minority of mostly young demonstrators torching cars and bin containers and launching petrol bombs at police.
Demonstrators have frequently cut road and rail links between Spain and France, prompting complaints from businesses, which say they must use longer routes to ship their goods abroad.
Many shops in downtown Barcelona have been shut during the rallies and there are growing concerns about the impact of the unrest on business in Spain's second largest city and its tourism capital.
Sunday's national election, Spain's fourth in four years, produced a divided parliament, setting the stage for a difficult negotiation on forming a government.