The Badger Trust had attacked Mr Justice Ouseley's decision in July to uphold government proposals for two pilot culls to tackle tuberculosis in cattle: one in West Gloucestershire and the other in West Somerset.
The Trust says that killing badgers will make no meaningful contribution to tackling the disease, which has been described as the most pressing animal health problem in the UK.
It claims that the scheme could lead to 40,000 animals being "pointlessly killed" over the next four years.
But, Lord Justice Laws, Lord Justice Rimer and Lord Justice Sullivan unanimously rejected the appeal, which was only concerned with statutory construction.
Afterwards, Patricia Hayden, vice-chairman of the Badger Trust said: "We are very disappointed. We don't know what the next step is, but we will not give up."
David Wolfe QC, for the trust, claimed the two culls would involve killing an estimated 3,400 badgers in each area - each approximately the
size of the Isle of Wight - and the long-term intention was to issue licences for up to 10 culls each year.
The licences for the culls, due to start later this autumn, would be issued under the Protection of Badgers Act 1992, which consolidated provisions from the 1973 Badgers Act.
He said that, in the 1973 Act, parliament put in place a regime of badger protection with limited exceptions, and it was inconceivable that it was providing a power to undertake widespread culling.
After the July ruling, which was welcomed by the the National Farmers Union, the British Veterinary Association and British Cattle Veterinary Association, a spokesman for the Department of Environment, Food and Rural Affairs (Defra) said: "No-one wants to cull badgers but last year bovine TB led to the slaughter of over 26,000 cattle, and to help eradicate the disease it needs to be tackled in badgers."
Cost of the cattle losses was estimated at £91 million.
Music legend Brian May said the fight against culling badgers would continue despite the defeat in the courts.
The Queen star said: "What you've heard is that the judicial review failed. It isn't totally unexpected.
"What you've got to remember is that judges do not review the scientific evidence, they do not review the ethical considerations. All they do is look at the technicalities.
"The issue was did the Government break any procedural rules? It was a good thing to try and I am not actually very much daunted by the fact it failed."
Mr May was speaking at a rally in Bristol to launch a new national campaign called Stop the Cull.
The campaign is a united response by leading animal welfare charities opposed to the proposed badger cull, which is due to take place in Somerset and Gloucestershire later this month.
The coalition consists of the RSPCA, League Against Cruel Sports, Save Me and Humane Society International (UK).
Mr May said that a legal challenge in Wales also failed in the courts but a badger cull was later scrapped and replaced with a vaccination programme.
"But what happened next was, when the cull was just about to go ahead, the Government changed and there was enormous public reaction to the fact they were going to kill badgers and Elin Jones (minister for rural affairs) was taken out of office probably partly because of that,"
the guitarist said.
"So things can change very fast. David Cameron has already reshuffled his cabinet.
"We know longer have the farming minister Jim Paice, we no longer have Caroline Spelman.
"We have a new team and I think we are all hoping that David Cameron is looking at the possibility of reorganising and delaying the cull while they look more carefully at vaccination."
Mr May said that the EU would not allow vaccination and he was campaigning for that to change.
"What I would like to do, if this cull can be shelved, I myself would volunteer to go in with the farmers to Europe to work on them to try to get permission to vaccinate our cows," he said.
"I can open doors, I'm a rock star. It's unorthodox but it works.
"Its a terrible tragedy if it goes ahead and its an irreversible tragedy - you can never bring those badgers back.
"You could kill all the badgers in Britain and it would not stop the problem of bovine TB in cows.
"All I can say is be with us, support TeamBadger.org, sign the petition and try to get David Cameron to reconsider and let's just pray we can succeed."
Badger Trust finance director Jeff Hayden - Patricia Hayden's husband
- added outside court: "A cull is not likely to work and it could make matters worse.
"Natural England have said as many as 130,000 badgers could be killed."
Mr Hayden warned that badgers might be shot and left injured and other animals might be hit.
He added police may have to deal with protesters.
"There may be protests. We have heard rumours," he added. "But the Badger Trust would not condone any illegal activity."
He urged ministers to explore the possibilities of vaccinating cattle and said trust officials were hoping to have discussions with new
Environment Secretary Owen Paterson, who replaced Caroline Spelman in a
Cabinet reshuffle last week.
Officials would discuss further possible steps with lawyers, Mr Hayden added.
"We remain hopeful. Certainly hopeful," he added. "Every bit of research that comes out seems to be in our favour."
PA
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