Recent studies have demonstrated that klotho protein plays a role in calcium/phosphate homeostasis. The goal of the present study was to investigate the regulation of Na-P(i) cotransporters in klotho mutant (kl/kl) mice. The kl/kl mice displayed hyperphosphatemia, high plasma 1,25(OH)(2)D(3) levels, increased activity of the renal and intestinal sodium-dependent P(i) cotransporters, and increased levels of the type IIa, type IIb, and type IIc transporter proteins compared with wild-type mice. Interestingly, transcript levels of the type IIa/type IIc transporter mRNA abundance, but not transcripts levels of type IIb transporter mRNA, were markedly decreased in kl/kl mice compared with wild-type mice. Furthermore, plasma fibroblast growth factor 23 (FGF23) levels were 150-fold higher in kl/kl mice than in wild-type mice. Feeding of a low-P(i) diet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. Finally, feeding of a low-P(i) diet resulted in increased type IIa Na-P(i) cotransporter protein in the apical membrane in the wild-type mice, but not in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type IIa/IIc transporters rather than to intestinal P(i) uptake.