"We expect at least 10,000 to attend, and hope that there will be at least 20,000," Axel Wieland, a spokesman for the organisers, told news agency AFP. The demo was due to start at 6:30 pm.
A previous protest on August 13 attracted "significantly" more than 20,000 people, organisers said. Police put the number of demonstrators at between 15,000 and 18,000.
The massive €4.5-billion Stuttgart 21 project aims to transform the southwestern city and the surrounding region into an important railway crossroads of 21st-century Europe.
Engineers plan to blast 16 tunnels and cuttings into the many surrounding hills, build 18 new bridges, lay 60 kilometres (38 miles) of new train track and create three new stations.
Stuttgart's main station will be utterly transformed, from a terminus into an underground through-station, so that trains no longer have to chug in and back
out but can whiz through on their way to Paris, Bratislava, Hamburg or Rome.
But many in Stuttgart say the project is far too disruptive and expensive and that the rail network could be speeded up in other, cheaper ways. They also fear it will go over budget.
In particular they object to the side wings of their historic train station building, an interwar modernist classic designed by Paul Bonatz, falling victim to the wrecking ball.
The traditionally conservative state of Baden-Württemberg, where Stuttgart is the capital, will hold elections in March, and some observers believe the unpopular rail project could affect support for Chancellor Angela Merkel's Christian Democrats.