To survive in hostile environments, organisms activate stress-responsive transcriptional regulators that coordinated increase production of protective factors. Hypoxia changes cellular metabolism and thus activates redox-sensitive as well as oxygen-dependent signal transducers. We demonstrate that Sirtuin 1 (Sirt1), a redox-sensing deacetylase, selectively stimulates activity of the transcription factor hypoxia-inducible factor 2 alpha (HIF-2α) during hypoxia. The effect of Sirt1 on HIF-2α required direct interaction of the proteins and intact deacetylase activity of Sirt1. Select lysine residues in HIF-2α that are acetylated during hypoxia confer repression of Sirt1 augmentation by small-molecule inhibitors. In cultured cells and mice, decreasing or increasing Sirt1 activity or levels affected expression of the HIF-2α target gene erythropoietin accordingly. Thus, Sirt1 promotes HIF-2 signaling during hypoxia and likely other environmental stresses.