The test route for the self-driving machine is in Bad Birnbach, set in the rolling hills of the southeastern state Bavaria not far from the Czech and Austrian borders.
Made by French startup EasyMile, the 12-person bus will offer free rides on an eight-minute route linking the baths, the town centre and the station, Deutsche Bahn (DB) said in a statement.
"We've just driven autonomously into a new era of transport," DB boss Richard Lutz, who rode along on the first trip, said in a statement.
The rail operator has launched a subsidiary dubbed Ioki to test future modes of transport, focusing especially on electric-powered mobility.
From 2018, the new buses will operate on test routes in several German towns, including the country's second-largest city Hamburg.
DB hopes that in the future they will operate like a private car service, picking up passengers from home on demand and bringing them to the station - picking up others with the same destination along the way.
Across the Americas, Asia and Europe, a number of cities - including Paris, Lyon, Las Vegas and Dubai - are already experimenting on a small scale with autonomous vehicles complementing public transport systems.