The trio from the group Robin Wood had occupied the giant mechanical digger for five hours, delaying the demolition work on the historic Stuttgart train station.
The operation went smoothly and peacefully, a police spokesman said. The protestors were taken into custody and charged.
The earth-mover is being used to demolish the north wing of the old train station.
Tens of thousands of Stuttgarters have demonstrated over the past 10 days against the project.
The €4.1 billion Stuttgart 21 project aims to transform the city into an important European rail hub, making the city of 600,000 a key station on one of the longest high-speed lines in Europe, the 1,500-kilometre (930-mile) "Magistrale for Europe" linking Paris, Strasbourg, Munich, Vienna and Bratislava, and Budapest.
Engineers plan to blast 16 tunnels and cuttings into the many surrounding hills, build 18 new bridges, lay 60 kilometres (40 miles) of new train track and create three new stations.
But opponents are furious that the project will, among other things, tear down the side wings of the train station building, an interwar modernist classic designed by Paul Bonatz.
Deutsche Bahn CEO Rüdiger Grube proposed on Saturday a roundtable between opponents and supporters of Stuttgart's controversial railway construction. He acknowledged communication about the project had been poor and insisted, "we need to start acting like grown-ups.”