The demonstration in the Tarn region against the proposed A69 motorway drew in 8,200 protesters according to organisers; 4,500 according to the local authority.
They marched under intermittent rain along the route of the proposed highway which would link the southern cities of Toulouse and Castres, carrying placards that read "Less energy, fewer cars and less tarmac" and other green messages.
Julien Bayou, a lawmaker from the Europe Ecology Greens party (EELV), said the planned highway was "anachronistic".
Sandrine Rousseau, another Greens MP, said the project dated back to the 1990s when the thinking was that one could only travel by cars and on highways.
"There is really no need of another motorway," she said.
Others said the 53-kilometre (30-mile) highway would lead to a loss of farm land and endanger biodiversity.
Last month, protests against planned water storage facilities in another southern region led to clashes between demonstrators and police in which three people were seriously wounded.
Campaigners in Sainte-Soline were trying to stop the construction of giant water "basins" to irrigate crops, which they say will distort access to water amid drought conditions.
The water reserve is the second of 16 such installations, part of a project developed by a group of 400 farmers to significantly reduce mains water usage in summer.
