Clashes have left 38 people dead in protests against moves to strengthen President Nicolas Maduro's hold on power and his refusal to hold early elections.
The center-right opposition blames socialist leader Maduro for an economic crisis that has caused severe shortages of food and medicine.
'Resistance' or 'insurgency'?
A senior opposition lawmaker, Freddy Guevara, told reporters Monday's rally would go on for 12 hours until 2300 GMT.
He said 50 other similar blockages were planned on roads across the country, with the aim of generating a state of "ungovernability" to pressure Maduro.
"This is a day of resistance, of endurance, of showing that we are organized and firm," Guevara said at the rally.
He repeated the opposition's claim that Maduro was installing a "dictatorship."
Maduro has vowed general elections will take place as scheduled in late 2018 but not before.
He has accused the opposition of mounting an "armed insurgency" and "terrorist acts" against him with US backing.
'Keep protesting'
Maduro has set no date for postponed regional polls or for mayoral elections that are due this year.
The opposition has called on the armed forces to abandon Maduro but so far he still has the public backing of the high command.
Analysts say street protests are one of the few means the opposition has left to pressure Maduro.
"There is no freedom. They repress us. There is no food and when there is it is extremely expensive," said teacher Miguel Martinez, protesting on Monday.
"I am going to keep protesting in the streets until there is a change."
The government and the opposition have accused each other of sending armed groups to sow violence in the protests.
Police have fired tear gas and protesters have hurled rocks and Molotov cocktails in a near-daily series of clashes.
History of coups
Venezuela has seen three attempted military coups since 1992.
Clashes at anti-government protests in 2014 left 43 people dead.
The current unrest erupted on April 1 after the Supreme Court seized power from the National Assembly legislature -- the only state authority controlled by the opposition.
The court reversed that move after an international outcry. But Maduro further fanned protests when he announced plans to reform the constitution.
That was seen by his opponents as a bid to dodge elections and cling to power.
The European Union on Monday urged all sides in Venezuela to refrain from violence and seek a political solution.
More about: #Venezuela
