The train operator introduced a trial service from London to Lyon in 2013, announcing it would make it a permanent feature earlier this year.
Corporate groups can now travel direct from London's St Pancras International or Ashford International in Kent to the French capital of gastronomy in just over four and a half hours.
After Lyon the train goes on to stop at Avignon in the Provence region and the coastal city of Marseille – a journey of just over six hours.
Nick Mercer, commercial director of Eurostar, said: "We’re delighted to send off our very first Eurostar passengers to Lyon and the South of France today, and with strong advance ticket sales, we look forward to welcoming many more in the coming months. Our new service brings the gastronomy, culture and art of the regions closer to the UK, with an effortless journey available all year round."
Passengers travelling from France back to the UK will be able to board their train without the need to arrive 30 minutes before departure, because check-in, security and immigration checks will take place at Lille Europe, where customers leave the train before continuing the journey to Ashford and London.
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