AbstractKlotho controls renal electrolyte handling by modulating tubular reabsorption of calcium and phosphate through the epithelial calcium channel TRPV5 and sodium phosphate co‐transporter NPT2A. The Ksp‐KL−/− mice have a targeted deletion of Klotho in the distal part of the nephron. Considering that the distal convoluted tubule is the most important site for Ca2+ regulation in the kidney, Ksp‐KL−/− mice were challenged with a Ca2+‐deficient diet for determining the Ca2+ handling and pinpointing the Klotho levels needed for controlling renal Ca2+ handling. The Ksp‐KL−/− mice displayed normal weight and showed unaltered calcium and phosphate levels in serum and 24‐h urine. Expression of calciotropic (Trpv5, Trpv6) and phosphotropic (Slc34a1, Slc34a2) genes in the kidneys, duodenum, ileum, and colon were not affected by Klotho deletion. In conclusion, our study reports that mice with 18%–93% residual levels of Klotho in the kidney exhibit normal electrolyte homeostasis when placed on a low Ca2+‐content diet.