Bosses at the Border Collie Trust (BCT), at Colton near Rugeley, say they will be forced to move before work starts on the high-speed network.
The trust, which bought the 4.5-acre countryside site for £200,000 back in 1997 – and has forked out £800,000 to develop it – will be forced to look for a new home.
An appeal has now been launched to raise money for the move.
The trust rehomes between 400 and 500 dogs each year.
Trustee Ben Wilkes, who has been involved in the centre since it opened, said: “We’ve been here for 21 years – it was an old boarding kennels
“We have spent a lot of time, money and effort in rebuilding and setting an ideal situation for a specific breed of dog that is often very nervous, wary and noise sensitive.
“When HS2 was first announced, it was a bit of a concern.
“We were only going to be affected because we were next to the line. It wasn’t directly taking any property.”
That changed when revised plans for HS2 were revealed last year.
Mr Wilkes explained: “The plans came out and it comes to light that Moor Lane is being rerouted and will come through our paddock.
“Not only we will lose 36 per cent of our land – but that road is going to be a construction route for three years, making it impossible to exercise the dogs.
“After a period of consideration, we had to make the decision that we can’t operate from here.”
The BCT, which employs around 13 local people, is in the process of submitting a Blight Notice, which requests HS2 to consider buying the land and property because it is not feasible for it to continue.
Negotiations will take place if the notice is given the go-ahead, but it is not certain that HS2 bosses will accept – leaving the trust in financial limbo.
Mr Wilkes, who lives at the centre, says it is important that the BCT remains local.
“They will either agree to the notice, then we get down to negotiating, or they refuse. That really would be the killer blow.
“We’ve got to find somewhere suitable without losing the staff and experience. We’ve set up an online donation platform. Even if they give us a fair price for the property, there are still costs we will have to take into account.”
Between 1999 and 2006, the trust built a 40- kennel rescue block, a 14-kennel boarding block, a special needs area and an office.
The BCT hopes to submit the Blight Notice to HS2 by the end of this month.
An HS2 spokesperson said: “We recognise that people are concerned about the impacts of construction, and we continue to engage with people and businesses to look at how we minimise impacts as we progress to the next stage of design.
"We have visited Border Collie Trust and met with its representatives to look at the impact on their business and listen to their concerns.
"We also recognise that every situation is unique.
"That’s why we have set up a package of property schemes including property compensation and assistance measures, to assist affected landowners.
"We are committed to ensuring that support is in place for individuals and communities directly affected by HS2.
"Delivering HS2 Phase 2a between the West Midlands and Crewe six years earlier than originally planned will improve journey times to northern cities sooner and support growth and jobs in the local area."
To find out more about the project or to donate to the fund go to bordercollietrustgb.homestead.com