Lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), is widely used for the treatment of mood disorders. Side effects of lithium include nephrogenic diabetes insipidus, leading to renal water loss. Dehydration has in turn been shown to downregulate Klotho, which is required as co-receptor for the downregulation of 1,25(OH)₂D₃ formation by fibroblast growth factor 23 (FGF23). FGF23 decreases and 1,25(OH)₂D₃ stimulates renal tubular phosphate reabsorption. The present study explored whether lithium influences renal Klotho expression, FGF23 serum levels, 1,25(OH)₂D₃ formation, and renal phosphate excretion. To this end, mice were analyzed after a 14-day period of sham treatment or of treatment with lithium (200 mg/kg/day subcutaneously). Serum antidiuretic hormone (ADH), FGF23, and 1,25(OH)₂D₃ concentrations were determined by ELISA or EIA, renal Klotho protein abundance and GSK3 phosphorylation were analyzed by Western blotting, and serum phosphate and calcium concentration by photometry. Lithium treatment significantly increased renal GSK3 phosphorylation, enhanced serum ADH and FGF23 concentrations, downregulated renal Klotho expression, stimulated renal calcium and phosphate excretion, and decreased serum 1,25(OH)₂D₃ and phosphate concentrations. In conclusion, lithium treatment upregulates FGF23 formation, an effect paralleled by substantial decrease of serum 1,25(OH)₂D₃, and phosphate concentrations and thus possibly affecting tissue calcification.