Klotho, a transmembrane protein, protease and hormone mainly expressed in renal tissue counteracts ageing thus leading to increase of life span. Overexpression of Klotho substantially prolongs the life span. Klotho-deficiency leads to excessive formation of 1,25(OH)₂D₃, growth deficit, accelerated aging and early death. Ageing is frequently paralleled by dehydration which is considered to accelerate the development of age related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR and Klotho protein abundance were detected by western blotting in renal tissue from hydrated and 36 hours dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase of plasma osmolarity as well as plasma ADH, aldosterone and 1,25(OH)₂D₃ levels. ADH (50 nM) and aldosterone (1 mM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.