More than 50,000 people took part in the rally in Minsk, according to the Viasna human rights centre.
Demonstrators carried banners mocking President Alexander Lukashenko, who has run the country for 26 years, and chanted “Go away!”
Mass protests have rocked Belarus since the August 9th presidential election that handed Mr Lukashenko a victory with 80 per cent of the vote. His main challenger, Sviatlana Tsikhanouskaya, got 10 per cent.
She and her supporters refused to recognise the results, saying the outcome of the vote was manipulated.
Authorities tried to quell the unrest with mass detentions and police dispersing crowds with truncheons, stun grenades and water cannons.
On Monday, the country’s Interior Ministry threatened to use firearms against the protesters “if need be”, saying that the rallies “have become organised and extremely radical.” But the protests have continued despite the crackdown.
“This is the first march since the authorities threatened to use firearms. But even that didn’t stop the protest, which transforms into different forms, but doesn’t die down,” Viasna leader Ales Bialiatski said.
Ms Tsikhanouskaya, who is in exile in Lithuania after leaving the country in fear for her safety, threatened on Tuesday to call a nationwide strike unless Mr Lukashenko announced his resignation, released political prisoners and stopped the crackdown on protesters.
“If our demands aren’t fulfilled by October 25th, the entire country will peacefully take to the streets,” Ms Tsikhanouskaya said in a statement.
“On October 26th, a national strike of all enterprises will begin, all roads will be blocked, sales in state-owned stores will collapse.”
Protesters marched down one of Minsk’s main avenues on Sunday, where plants and factories are located, and chanted “strike” and “the workers are with the people”.
The city centre was blocked off by water cannons and armoured vehicles, and dozens of military trucks were seen driving around the streets.
Several underground stations were closed, and mobile internet was not working in the area where the march was happening.
“Lukashenko understands that if there hadn’t been all these artificial obstacles, the entire Minsk would have come out (to rally),” Valery Karbalevich, an independent political analyst in Minsk, said.
In a statement on Sunday, Ms Tsikhanouskaya encouraged Belarusians to continue peaceful protests.
“Let’s continue to express our demands peacefully and persistently — it is yielding results,” she said, adding that the authorities had released Ilya Salei, the lawyer of Ms Tsikhanouskaya’s top associate, Maria Kolesnikova, from detention.
Ms Kolesnikova was jailed last month on charges of undermining state security that could bring a five-year prison term if she was convicted. Mr Salei was detained in September as well in relation to the same charge.
Protests took place in several big cities apart from Minsk on Sunday, including Brest, Grodno, Gomel and Vitebsk.
Scores of demonstrators have been detained across the country: the list of detained protesters released by the Viasna centre on Sunday evening had more than 150 names on it.
