Hypoxia inhibits Na-K-ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase. We found that in the presence of pVHL hypoxia decreased Na-K-ATPase activity and promoted the degradation of plasma membrane Na-K-ATPase. In pVHL-deficient cells, hypoxia did not decrease the Na-K-ATPase activity and the degradation of plasma membrane Na-K-ATPase was prevented. pVHL-mediated degradation of Na-K-ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant-negative Ubc5 prevented Na-K-ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL-mediated Na-K-ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2, did not affect the half-life of plasma membrane Na-K-ATPase. In addition, stabilizing HIF1/2 by infecting mammalian cells with adenoviruses containing the oxygen-dependent degradation domain of HIF1 did not affect the plasma membrane Na-K-ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na-K-ATPase was not degraded during hypoxia, whereas cells with knockdown of HIF1/2 retained the ability to degrade plasma membrane Na-K-ATPase. These findings suggest that pVHL participates in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase in a HIF-independent manner.—Zhou, G., Dada, L. A., Chandel, N. S., Iwai, K., Lecuona, E., Ciechanover, A., Sznajder, J. I. Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein.