Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as co-substrates with iron (II) and vitamin C serving as co-factors. While vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here we report that vitamin C deprived Gulo(-/-) knock-out mice show normal HIF-dependent gene expression. The systemic response of Gulo(-/-) animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all three PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity and endogenous target gene expression in cells exposed to CoCl(2). A C201S mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.