Activists from the ‘Robin Hood' and ‘Parkschützer' (park protectors) environmental groups also occupied several trees in the central Schlossgarten park where around 300 trees are set to be cut down.
Work has reportedly begun at the nearby disused central bus station, with bore holes being drilled in preparation for work to sink the ground water level ahead of the construction of the underground train station.
The current central train station will become the above-ground part of a huge underground station, which trains will pass through according to the plan, set to be completed by 2019.
Stuttgart police estimated the crowd number to be around 30,000 - although organisers said they reckoned 65,000 people had shown up to protest during the evening.
A meeting between representatives of Stuttgart 21 and those against the project will take place on September 10, it was confirmed by Baden Württemberg state authorities on Friday.
State premier Stefan Mappus, of the Christian Democratic Union, and head of the Green Party state parliamentary group, Winfried Kretschmann, had previously agreed to meet without any preconditions.
But those hoping to stop the project are still working to try to get demolition work halted while the talks are being held.
Political support for a limited halt to the work is growing, with Social Democratic Party state leader Nils Schmid calling for such a move, and two city Christian Democratic Union politicians also putting their weight behind the idea.