Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (approximately 190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.