Head coach Gareth Jenkins made the "tough call" after deciding Henson, whose career has been stalled by a string of injuries, had run out of time to reach peak form and fitness.
As a result, the former British Lion was omitted from the Wales squad for Saturday's Test against England at Twickenham - the last match before Jenkins' has to name his 30-man World Cup squad.
"We have taken an early decision with Gavin and decided that he is no longer in contention for the World Cup squad which must be named before August 14 and so have not considered him for selection against England," Jenkins explained.
"We have given him every opportunity to make it and it's been a tough summer for Gav in which he has worked hard.
"But the recent Achilles injury in particular has meant he's not been able to train at full pelt and that has reflected in his conditioning. I think we all agree he is not where he needs to be."
Four years ago Henson was left "bitter and hurt" by his omission from Wales' World Cup squad - but on this occasion the circumstances were very different.
While Henson had considered himself "the best player in Wales" in 2003 when he was overlooked by Steve Hansen, this was a call he sensed was coming.
Henson has started just one Test under Jenkins - the 29-29 draw with Australia in November - and endured another stop-start season due to injuries.
Henson said: "Its extremely disappointing for me as it is every player's dream to play rugby for their country and I've taken great pride in pulling on the Wales every time I've been selected.
"It would've been particularly special to do so at the World Cup. I can understand the decision that's been taken. I know I'm not quite where I would like to be at the moment conditioning wise.
"It's been frustrating. I've had to play catch up for a while and these games have come a bit too early for me."
Henson expects to reach full fitness by September and Jenkins confirmed he will remain in contention for a late call-up if the squad is further hit by injuries.
Wales are already without key number eight Ryan Jones and Jenkins is sweating over fly-half Stephen Jones, who is out of the England game with a torn groin muscle.