Low atmospheric pressures on Mars and the lack of substantial amounts of liquid water were suggested to be among the major limiting factors for the potential Martian biosphere. However, large amounts of ice were detected in the relatively shallow subsurface layers of Mars by the Odyssey Mission and when ice sublimates the water vapour can diffuse through the porous surface layer of the soil. Here we studied the possibility for the active growth of microorganisms in such a vapour diffusion layer. Our results showed the possibility of metabolism and the reproduction of non-extremophile terrestrial microorganisms (Vibrio sp.) under very low (0.01–0.1 mbar) atmospheric pressures in a Martian-like shallow subsurface regolith.