Stagecoach will provide a fleet of 150 double-decker buses, which will run to and from Ryder Cup park-and-ride sites at Perth, Stirling and Kinross. The company previously ran buses for the 2010 Ryder Cup in Wales.
Ryder Cup Europe, which awarded the contract, declined to comment on the value of it, as did Stagecoach. But it is understood to be worth more than £500,000.
Richard Hills, European Ryder Cup director, said: "It is a huge operation to transport in the region of 45,000 people to and from the golf course every day during the week of the 2014 Ryder Cup.
"Visitors to the event will be able to enjoy a comfortable and pleasurable journey from their park-and-ride base."
He added: "We are delighted that Stagecoach, a company with its roots firmly in Scotland, will facilitate these transfers and ensure the smooth arrival and departure of the spectators at Gleneagles."
The bulk of the operation will be managed by Stagecoach East Scotland, based at Kirkcaldy in Fife. Vehicles will also be provided by Stagecoach's regional bus companies across the UK.
Robert Montgomery, managing director of Stagecoach's UK bus division, said: "We're delighted to be involved in such a prestigious, global sporting event, which has not been held in Scotland since 1973."
Ryder Cup Europe said that Stagecoach had delivered a "successful" transport operation at The 2010 Ryder Cup in Wales.