This will be first time the endangered mammals have been seen in England’s highest freshwater lake for 50 years.
The water voles will be released in the fen area of the Tarn this year, says the Trust, with a further hundred voles released in June 2017.
Bred in captivity, the reintroduction is part of the Trust's major new vision for land management in the Yorkshire Dales.
The water vole is Britain’s fastest declining wild mammal, with numbers falling by almost 90% in recent years, and National Trust ecologists believe Malham Tarn’s water voles were wiped out in the 1960s by mink, which escaped from fur farms nearby.
According to the National Trust, the water voles will play an important part in the ecosystem at Malham Tarn, grazing and burrowing into areas of the riverbank and providing the space for rare plants like scarce mosses and liverworts that need patches of open habitat to grow. The water voles will also act as a food source for struggling predators like barn owls and otters.
Roisin Black, National Trust Ranger at Malham Tarn, said: “In the rest of Europe, water voles are common. In Britain, the creatures are incredibly rare. We know water voles have thrived at Malham Tarn in the past and thanks to work by the National Trust, the habitat here is perfect for water voles again.
“By reintroducing water voles to the Tarn, we hope to give these rare animals the chance to recolonise the streams in the high Yorkshire Dales.”