The project, which aims to use cyanide to mine 314 tonnes of gold and 1,500 tonnes of silver, has drawn fierce opposition from civic rights groups and environmentalists, who say it would destroy ancient Roman gold mines and villages.
It is led by Rosia Montana Gold Corporation, majority-owned by Canada's Gabriel Resources Ltd with the Romanian government holding roughly 20 percent.
The project has been valued at $7.5 billion based on a 2007 study that used an average price of $900 per ounce of gold, with Romania estimated to get about 75 percent of the benefits in taxes, royalties, dividends and jobs.
Gold currently trades around $1,390 per ounce.
In the capital Bucharest, up to 3,000 protesters marched toward the government headquarters from University Square, the scene of violent anti-austerity protests early last year that toppled a previous government.
Protesters held aloft banners saying "United for Rosia Montana" and "Our children don't want cyanide." Protesters also gathered in the northwestern Romanian city of Cluj. A separate rally against shale gas exploration, drew another 2,000 people onto the streets in the eastern town of Barlad.
"This protest can get people together," said Ioana Paun, 28, who joined the protest in Bucharest. "From what I am hearing around me, this is only the beginning."
Prime Minister Victor Ponta strongly opposed the project before he took power in May last year yet voted for the draft law enabling the mine, only to tell a local television station that he would vote against the project in parliament.
Most Rosia Montana residents hope the project will bring jobs and money to their impoverished town, which suffered when a state-owned gold mine closed in 2006. Only a small number of the town's of 2,800 residents refuse to sell their property to make way for the mine.
The company proposes carving open four quarries over the mine's lifespan, work that would destroy four mountain tops and wipe out three outlying villages of the 16 that make up Rosia Montana municipality, while preserving the town's historical center.