When exposed to hypoxia (1.5% O2), several cell types have been shown to increase production of reactive O2 species derived from the mitochondrial electron transport chain (mtROS). The general physiological consequences of hypoxic mtROS production are not completely understood, although several groups have demonstrated that mtROS promote the stabilization and activity of hypoxia inducible factor-1alpha (HIF-1alpha) transcription factor, alter cardiac myocyte contractility, and modulate Na+-K+-ATPase activity. To investigate the effects of hypoxia-induced mtROS on general cellular oxidative metabolism, we measured the levels of glutathione, a major cellular antioxidant, in response to hypoxic treatment. Our data indicate that HEK293 and Hep3B cells exposed to 1.5% O2 exhibit a time-dependent decrease in cellular glutathione stores and concomitant inhibition of glutathione biosynthesis, which correlates to impaired transport of the substrate cystine. Using a combination of ROS scavengers, mitochondrial electron transport inhibitors, and mitochondrial DNA-deficient rho0 cells, we demonstrate that this decrease in cellular glutathione levels is mediated by hypoxia-induced mtROS. Intriguingly, this effect is also inhibited by cyclohexamide but is not dependent on HIF-mediated transcription. Overall, these data suggest a novel HIF-independent role for mitochondrial ROS in regulating glutathione synthesis, and hence cellular oxidative homeostasis, during hypoxic exposure.