The roadblocks have caused widespread disruption in all Brazilian states.
The Senate is due to vote on the impeachment on Wednesday. A simple majority in favour of a full trial will suspend Ms Rousseff for up to 180 days.
The government has asked the Supreme Court to suspend the impeachment process for alleged irregularities.
However, similar attempts have been rejected by the court.
Protesters blocked the main access roads to Brazil's largest city, Sao Paulo, causing huge traffic jams during the morning rush-hour.
The protests spread to cities and towns across the country.
Police said there were barricades on 14 highways in Brazil's 26 states and the Federal District, home to the capital Brasilia.
In Rio de Janeiro, which will host the Olympics in August and September, protesters set up barricades at a major road linking the city to the state of Sao Paulo.
In the north-eastern city of Natal, buses did not run in the morning and the road next to the main university was blocked. Classes were suspended.
The anti-impeachment protests were organised by a left-wing umbrella organisation, the Popular Brazil Front, which promised to carry on with its direct action tactics.
"We will fight everywhere to defend democracy. Brazil says no to the coup!" it said in a Facebook message.
President Rousseff has vowed to keep fighting until the end against her impeachment.
On Tuesday, the acting Speaker of the lower house of Congress, Waldir Maranhao, surprised the country when he announced that he had suspended the vote that allowed the impeachment process against her to go ahead.
He said the 17 April vote in the lower house had breached Congress rules. Members had voted overwhelmingly in favour of the impeachment process going ahead.
But within less than 24 hours he had revoked his decision to annul the vote. He gave no reason for his u-turn.
If the Senate votes goes ahead on Wednesday and Ms Rousseff loses it, she will be replaced by Vice-President Michel Temer while the trial lasts.
She says Mr Temer is a traitor who is taking part in a political coup against her democratically elected government.
The president is accused of illegally manipulating finances to hide a growing public deficit ahead of her re-election in 2014, which she denies.
Supporters of Brazil's President Dilma Rousseff have set up burning barricades and blocked roads across the country in protest against moves to impeach her.