The Swansea-Cork ferry service is to close with the loss of 78 jobs.
It was suspended in November due to "rising fuel costs", but backers hoped to resume services in March.
The owners, West Cork Tourism Co-operative Society, said state aid rules and "red tape" prevented them at the 11th hour from re-launching the service despite pledges of financial support.
The Welsh Government described the news as disappointing, but insisted the rescue plan was not "commercially viable". It is now expected the ferry company which ran the service, Fastnet Line, will be placed into receivership or liquidation.
The owners said as well as direct job losses, the closure would cost 20 million euro in lost tourist spending in south Wales and about 30 million euro in south west Ireland.
Noel Murphy, chairman of the West Cork Tourism Co-Operative, said: "Despite heroic efforts by staff and supporters of the ferry service in both Ireland and Wales, we are very disappointed to announce that we could not to save this vital piece of tourism and transport infrastructure.
"Unfortunately, our efforts fell at the final hurdle. The funds were there, private and public, to allow us to continue but, despite the best efforts of all involved, state aid rules and red tape choked off the ferry's chances of sailing again in March 2012."
The Swansea to Cork Ferry Service was first established in 1987. However, it came to a halt in 2006 when the then operating firm Swansea Cork Ferries Ltd sold its vessel.
In 2010 the route was re-established - this time run by the co-operative Fastnet Line. But it ran into difficulty last year when it was announced the service was to be suspended over the winter months - blaming higher-than-expected fuel prices.
Fastnet Line was then entered into a process known as "interim examinership" by a court in Dublin - a recovery process allowed within Irish business law.