We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O₂ had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of α1 Na,K-ATPase protein decreased on exposure to 1.5% O₂ for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40°C and 1.5% O₂, the degradation of the α1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient ρ⁰ cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,K-ATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na⁺ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na,K-ATPase via the ubiquitin-conjugating system.