The inward rectifier K⁺ channel Kir2.1 contributes to the maintenance of the resting cell membrane potential in excitable cells. Loss of function mutations of KCNJ2 encoding Kir2.1 result in Andersen-Tawil syndrome, a disorder characterized by periodic paralysis, cardiac arrhythmia and dysmorphic features. The ubiquitously expressed protein kinase B (PKB/Akt) activates the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which in turn regulates a variety of carriers and channels. The present study explored whether PKB/PIKfyve contribute to the regulation of Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type PKB, constitutively active ^T308D,S473DPKB or inactive ^T308A,S473APKB. Kir2.1 activity was determined by two-electrode voltage-clamp. As a result, wild type PKB and ^T308D,S473DPKB, but not inactive ^T308A,S473APKB significantly increased Kir2.1-mediated currents. The effect of PKB was mimicked by coexpression of PIKfyve. The decay of Kir2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 mM) was similar in oocytes expressing Kir2.1 + PKB or Kir2.1 + PIKfyve as in oocytes expressing Kir2.1 alone, suggesting that PKB and PIKfyve influence channel insertion into rather than channel retrieval from the cell membrane. In conclusion, PKB and PIKfyve are novel regulators of Kir2.1.