Klotho inhibits 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Klotho-deficient mice (klotho^-/-) suffer from severe growth deficit, rapid ageing and early death, events largely reversed by vitamin D deficient diet. The present study explored the role of Klotho-deficiency on mineral and electrolyte metabolism. To this end, klotho^-/- mice and their wild type littermates (klotho^+/+) were subjected to normal diet (D⁺), Vitamin D deficient (D^-) diet or to vitamin D deficient diet for 4 weeks and then to normal diet (D^-/+). At the age of 8 weeks, body weight was significantly smaller in klotho^-/-D⁺ mice than in klotho^+/+D⁺ mice, klotho^-/-D^- and klotho^-/-D^-/+ mice. Plasma concentrations of 1,25(OH)₂D₃ and aldosterone were significantly higher, plasma PTH concentrations significantly lower in klotho^-/-D⁺ mice and klotho^-/-D^-/+ mice than in klotho^+/+D⁺ mice and klotho^-/-D^- mice. Plasma Ca²⁺, phosphate and bicarbonate concentrations tended to be higher and plasma K⁺ concentration tended to be lower in klotho^-/-D⁺ mice and klotho^-/-D^-/+ mice than in klotho^+/+D⁺ mice and klotho^-/-D^- mice. Urinary excretion of Ca²⁺, phosphate and K⁺ was significantly higher in klotho^-/-D^-/+ mice than in klotho^-/-D^- and in klotho^+/+D⁺ mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)₂D₃ in Klotho-deficient mice affects not only mineral but as well electrolyte metabolism.