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.

The two areas, which are currently car parks on Lord Street and Parade Street, occupy more than three acres.
Subject to planning, it is hoped the development will include leisure facilities, a hotel, shops and offices.
Infrastructure Minister Phil Gawne said potential designs need to "set the standard for future development".
He added: "This is an opportunity to develop a prime area in Douglas fronting the inner harbour and marina and close to the main shopping streets."

Starting and ending at the Town Hall in the city centre, the route took in views of the Peak District and a climb up to the village of Ringinglow.
The event saw shorter routes for junior runners, with the main 13.1-mile event won by Steven Bayton and Sarah Lowery.
Among the runners were retired rugby league players Barrie McDermott and Keith Senior.
Lowery, the first woman to finish the circuit, said: "I had not done this race before so it's nice to do your local half marathon.
"There were loads of people in the crowd all the way round, even in the really remote parts of the course, so I really enjoyed the race."
Councillor Mary Lea, from Sheffield City Council, said: "Although the route is undoubtedly a challenging one, the view from the top of Ringinglow Road across the Peak District is surely one of the best rewards any weary runner could hope for.
"I would go as far as to say that Sheffield is surely the most picturesque of almost any city half marathon, when you consider the blend of the beautiful rural landscape and the vibrant city centre finish."

The Supreme Court's judgement follows an appeal against the High Court's rejection of ministers' arguments.
Theresa May says the government already has powers to invoke Article 50 of the Lisbon Treaty - starting Brexit talks - by the end of March.
But campaigners say Parliament must be consulted before she does so.
Labour has already said it will not vote against the government over invoking Article 50.
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.
[NEXT_CONCEPT]
The Manx government has invited developers to come forward with ideas to transform prominent quayside sites in Douglas.
[NEXT_CONCEPT]
Thousands of runners have tackled the third Yorkshire Half Marathon under blue skies in Sheffield.
[NEXT_CONCEPT]
The government will learn next Tuesday whether it has won its legal battle to get Brexit under way without the need for a vote by MPs.