The secretary of state Owen Paterson now has to listen to the voice of Parliament and discard any plans to roll the cull out further.
MPs voted overwhelmingly to halt the controversial badger culls in England on Thursday, but the backbench motion does not bind ministers to abandon the policy.
The motion, proposed by the Conservative MP Anne Main, stated that the pilot culls had "decisively failed" and was passed by 219 votes to one. But farming minister George Eustice refused to respond to a challenge to hold a full debate and vote in government time.
The badger cull trials, aimed at curbing the rise of tuberculosis in cattle, failed to shoot the target number of badgers and also failed to meet humaneness criteria, according to leaked information about an independent report.
A setback for ministers
The vote represents a new setback for ministers, who also lost the only previous parliamentary vote on the policy in October 2012, which was prompted by a huge response to an official petition to stop the cull.
After the vote, shadow farming minister Huw Irranca-Davies told MPs: "Today Parliament has expressed its very clear view [against] the mass cull of badgers ... We have already the cross-bench support for a new way forward, a new consensus based on vaccination and cattle measures."
But Eustice defended the culls, saying: "Our view is there is nowhere, anywhere in the world, that has managed to successfully tackle TB without also dealing with the reservoir of the disease in the wildlife population."
Sympathy for farmers
The number of TB-infected cattle slaughtered in Wales each year, where no culling is taking place, has halved since 2008.
Earlier, Main told MPs: "There is great sympathy with farmers who have experienced heartache and hardship over losing cattle and precious stock to bovine TB.
"I believe many [MPs] lent their support to the concept of trying to tackle bovine TB with this strategy, but they did not give the government permission to carry on regardless: regardless of humaneness, regardless of effectiveness and regardless of costs."
Paterson must stop the cull
The RSPCA head of public affairs, David Bowles, said: "We are delighted the MPs have listened to the advice of the experts and voted overwhelmingly to abandon the badger cull.
"The secretary of state Owen Paterson now has to listen to the voice of Parliament and discard any plans to roll the cull out further."
"This is a huge disaster for the badger cull policy", said Dominic Dyer, chief executive of the Badger Trust. "Better farming practices, tighter biosecurity, better TB testing, more controlled cattle movements. Those are the answers to this disease, not badgers."
Damian Carrington is the head of environment at the Guardian.
This article is reproduced by kind permission of The Guardian.