Klotho, a transmembrane protein, protease and hormone, contributes to inhibition of 1,25(OH)₂D₃ formation and participates in the regulation of epithelial Ca²⁺, phosphate and electrolyte transport. Klotho deficiency leads to early appearance of age related disorders and premature death. The pleotropic disorders resulting from Klotho deficiency include cardiac arrhythmia and sudden cardiac death, pointing to deranged regulation of cardiac cell membrane potential. Deranged cardiac excitation may be at least in part due to excessive formation of 1,25(OH)₂D₃, but may involve in addition direct regulation of cardiac ion channels by Klotho. The resting potential of cardiomyocytes is maintained by inwardly rectifying K⁺ channels Kir2.1. The present study thus explored the possibility that Klotho regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected intoXenopus oocytes with and without additional injection of cRNA encoding Klotho. Kir2.1 activity was determined by two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, coexpression of Klotho significantly decreased Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Treatment of Kir2.1-expressing oocytes with Klotho protein was similarly followed by a gradual decline of Kir2.1 activity. In conclusion, Klotho is a novel regulator of Kir2.1.