Homozygous klotho mutant (kl-/-) mice exhibit multiple phenotypes resembling human aging. To elucidate the molecular basis of these singular phenotypes, we focused on the mechanisms underlying increased serum concentrations of calcium and phosphorus in kl-/- mice. Serum concentrations of calcitonin and PTH of kl-/- mice were normally up- and down-regulated, respectively, in response to the high levels of calcium. On the other hand, despite the high concentrations of calcium, serum levels of 1,25-dihydroxyvitamin D [1,25-(OH)2D] in kl-/- mice were significantly higher than that of wild type (WT). The expression of 25-hydroxyvitamin D 1alpha-hydroxylase gene, the key enzyme of vitamin D metabolism, was also greatly enhanced in kidneys of kl-/- mice. Furthermore, the normal genetic responses to administered 1,25-(OH)2D3, such as down-regulation of the 25-hydroxyvitamin D 1alpha-hydroxylase gene and up-regulation of 24-hydroxylase and VDR genes, were apparently impaired in kl-/- mice. These findings suggest that this deterioration in the vitamin D endocrine system may result in many of the phenotypes in kl-/- mice through effects of increased levels of calcium and phosphorus and 1,25-(OH)2D. Klotho protein may participate in calcium and phosphorus homeostasis via the regulation of the 1,25-(OH)2D signaling pathway.