Beanford Lane is closed to traffic to allow an annual toad migration
Wildlife experts are concerned hundreds of toads will die after cold weather prevented their migration.
A road into the Nottinghamshire village of Oxton is closed annually for one month to allow the safe passage of migrating toads.
Beanford Lane is a popular breeding area for about 1,000 amphibians as they return to their birthplace to mate.
But as the road re-opened on Wednesday, Nottinghamshire Wildlife Trust said many may now die in the weeks ahead.
Residents in the village became alarmed after years of "toad carnage" and campaigned to have the lane closed for the month of March.
The closure - which has been carried out for the past 10 years - is supported by the Nottinghamshire Wildlife Trust.
The charity said it also faced a funding shortfall which might mean next year's closure may not go ahead.
Erin McDaid from the trust said: "After 10 successful years we face a double blow. Not only has the weather thwarted our efforts to save toads this year - we now face a major funding shortfall making planning for future road closures very difficult."