Caffe Nero said it would stop using milk from farms in Somerset and Gloucestershire that were part of a pilot tackling badger numbers.
Their move follows action against the coffee shop chain on Facebook by anti-cull protesters.
A spokesman for the Stop the Cull group denied any threats being made.
The cull, in Gloucestershire and Somerset, was part of a government pilot aimed at killing 70% of the badgers to test how effective, humane and safe a cull could be.
Ministers believe killing badgers will curb TB in cattle, but opponents argue shooting is not the best way to eradicate the disease.
A spokesman for the Department for Environment, Food and Rural Affairs (Defra) said: "It is wholly unacceptable for a small group of protestors to intimidate and threaten retailers in this way.
"Our strategy for tackling bovine TB is based on advice from the Chief Veterinary Officer about the best way to control this harmful disease which threatens the future of our dairy and beef industries.
"We will continue to work closely with the dairy industry and retailers to offer them all the support we can."
Jay Tiernan, from Stop the Cull, denied there had been any threats against the cafes but said the chain had stopped selling the milk over animal rights issues.
He said it was "most likely" because of the "British Veterinary Association now speaking out [about] the method with which the badgers are shot".
Threats to a coffee shop chain for using cows milk from farms in the badger cull zone is unacceptable, the government has said.