Klotho, a protein mainly expressed in the kidney, counteracts ageing and thus increases the life span. Accordingly, the life span is by 80% shorter in mice with decreased klotho expression (klotho^hm) than in their wild type littermates (klotho^+/+). Disorders of klotho^hmmice include hypoglycemia resulting from enhanced insulin sensitivity of peripheral tissues. Klotho inhibits the 1a-hydroxylase and Klotho deficiency leads to excessive 1,25(OH)₂D₃ formation. The present study explored whether excessive formation of 1,25(OH)₂D₃ contributes to the deranged glucose metabolism. Experiments were performed in klotho^hm and klotho^+/+ mice, subjected to normal (D⁺) or Vitamin-D-deficient diet (D^-). Additional experiments were performed in C57Bl6 mice without (WT) or with (WT^1,25OH2D3) administration of 1,25(OH)₂D₃. At the age of 7 weeks, plasma 1,25(OH)₂D₃ concentrations were significantly higher and fasting plasma glucose and insulin concentrations were significantly lower in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice and klotho^hmD^- mice. An intraperitoneal glucose load (3 g/kg body weight) was followed by an increase of plasma glucose concentration, which was significantly less sustained in klotho^hmD⁺ mice than in klotho^+/+D⁺ mice and klotho^hmD^- mice, as well as significantly less sustained in WT^1,25OH2D3 mice than in WT mice. Intraperitoneal insulin (0.15 U/kg body weight) was followed by a decrease of plasma glucose concentration, which was less pronounced in klotho^+/+D⁺ mice and klotho^hmD^- than in klotho^hmD⁺ mice, as well as less pronounced in WT than in WT^1,25OH2D3 mice. In conclusion, the present observations disclose a role of klotho-sensitive 1,25(OH)₂D₃ formation in the regulation of cellular glucose uptake and insulin sensitivity.