Three brand new Falkirk-built buses each have space for two bikes each on the X62 route.
Two additional bike bays are to be added by the end of next month.
Borders Buses said each of the £275,000 buses could take up to 76 cars off the road.
Operations manager Lee Young said: “Active travel is increasingly becoming a popular choice with people from all walks of life.
"As a key transport provider, we are committed to continuously tailoring our product offerings to meet those changes in travel demand.
Scottish Borders Council leader Shona Haslam said: “The Scottish Borders is the leading cycling destination in Scotland, from the forests which provides a world-class mountain biking offering to the various cycling routes which allows people to explore our fantastic area.
“Tweeddale in particular has an excellent offering for cyclists of all ages and abilities, including the recent extension of the Tweed Valley multi-use path and opening of the e-Bike Hub in Innerleithen.
“The provision of these brand new bike-friendly vehicles Buses on the main service through Tweeddale will be hugely welcomed by local people and visitors and we hope will see even more people travel here.”
Charlie Miller, regional sales manager at Falkirk-based bus builders Alexander Dennis said it had worked with Borders Buses "to find this fantastic solution to carrying bikes on buses".
A spokesman for manufacturer said: "As far as we and Borders Buses are aware, these are the first in Scotland to carry bikes inside the vehicle.
"Other buses and coaches, when they do carry bikes, have external racks."
The vehicles also have free wi-fi, and wireless and USB charging points.