The inaugural journey on the weekday service left at 5:26am yesterday – it has been introduced as part of Virgin Trains’ proposals when it took over operations on the East Coast route in March.
Passengers now do not have to change trains in Edinburgh, cutting their journey times by around 46 minutes, Virgin said.
Business leaders, politicians and customers have welcomed the development.
Mr Mackay said: “I am pleased to launch this service from Stirling to London and I am confident that it will be a major draw for tourists, students, business and residents and deliver significant economic benefits to Stirling and the wider locality.
“It will bring further attention to Stirling’s diverse and growing business base, its splendid university and Business Innovation Park, its impressive cultural and sporting facilities and its historical attractions.”
The route is part of Virgin plans to increase services on the East Coast line with a fleet of 65 bigger, faster and more environmentally friendly trains from 2018, allowing it to provide more seats, accelerate journeys and attract more people to rail.
Warrick Dent, safety and operations director for Virgin Trains on the East Coast, said: “This is exciting day for us as we connect another of Scotland’s cities with a direct, early-morning service to London and start our journey to transform passenger services to Scotland.
“We’ve been really encouraged by the feedback from people in Stirling, particularly among businesses who have told us how much they value having this additional service, which will get them to and from London faster and without having to change trains.”
One of the passengers on board the first service was the Falkirk Scottish Nationalist MP, John McNally.
He said: “I usually get the train down to London on Sunday to get to Westminster. Having the early morning direct departure from Falkirk means travel to London on the Monday will be a much simpler option.”
VisitScotland chairman Mike Cantlay said: “This new direct service from London to Stirling is great news for Scottish tourism.
“Londoners will be able to escape the stresses of daily life to discover this beautiful historic city with its spectacular castle, cobbled streets and Renaissance churches and mansions.”