α-klotho was identified as a gene associated with premature aging–like phenotypes characterized by short lifespan. In mice, we found the molecular association of α-Klotho (α-Kl) and Na⁺,K⁺-adenosine triphosphatase (Na⁺,K⁺-ATPase) and provide evidence for an increase of abundance of Na⁺,K⁺-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca²⁺]_e) rapidly induce regulated parathyroid hormone (PTH) secretion in an α-Kl- and Na⁺,K⁺-ATPase–dependent manner. The increased Na⁺ gradient created by Na⁺,K⁺-ATPase activity might drive the transepithelial transport of Ca²⁺ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of α-Kl in the regulation of calcium metabolism.