Ahead of the rally, two leading protest figures said security officials had briefly detained them, warning them that any moves to block roads during the protest would be considered illegal.
Thousands chanted "Rio Tinto get out of Serbia" and "You won't dig" as they rallied in downtown Belgrade before setting off on a march through the city.
Interior Ministry figures later estimated the crowd size at between 24,000 and 27,000 people. Protestors later entered Belgrade's main railway station where demonstrators blocked tracks, halting traffic.
"The organisers and leaders were warned by the police both before and during the protest that their actions were not in accordance with the law," Interior Minister Ivica Dacic said in a statement, vowing charges would be filed against "all perpetrators".
Serbia has vast lithium deposits near the western city of Loznica. A mining project being developed by the Anglo-Australian mining giant Rio Tinto has been a perennial political fault line in the Balkan country in recent years because of its potential environmental impacts.
The deposits were discovered in 2004, but weeks of mass protests forced the government to halt the project in 2022.
The government revived the project following a court decision last month that said the order to revoke the permits awarded to Rio Tinto was "not in line with the constitution and the law".
The Serbian government signed a memorandum of understanding with the EU that is seen as the first step in developing Serbia's lithium resources.
- Protesters promise disruption -
Lithium is a strategically valuable metal needed for electric vehicle batteries, making it key for helping the automotive industry shift to greener production.
The project, however, has continued to be unpopular with many in Serbia due to concerns the mine would pollute water sources and endanger public health.
"I am in Belgrade because the survival of life in Serbia is being defended here," said Slobodan Stanimirovic, 58, from western's Serbia's Radjevina near the site of the future mine.
The Belgrade protest was the latest in a series held across the country after the mine's licences were reinstated.
Late Saturday, protest leader Zlatko Kokanovic promised to block more rail crossings in the coming days.
"We will stay here all night and all day," said Kokanovic.
Activists and demonstrators want legislators to pass a law permanently banning the mining of lithium and boron in Serbia.
Environmental groups said they were prepared to block major traffic arteries across Serbia and engage in civil disobedience if the government refused to act before an August 10 deadline that activists set.
Serbian President Aleksandar Vucic has repeatedly vowed that no mining operations will begin until guarantees over environmental safety protocols are established.
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