Abstract Aims: The signaling molecule hydrogen sulfide (H2S) protects cells against oxidative stress and activates NF-E2 p45-related factor 2 (Nrf2), a transcription factor that regulates antioxidant genes. We sought to establish whether H2S requires Nrf2 to protect against oxidative stress, and whether activation of Nrf2 by H2S involves antagonism of Kelch-like ECH-associated protein-1 (Keap1), a redox-sensitive ubiquitin ligase substrate adaptor that represses Nrf2 under normal homeostatic conditions. Results: H2S stabilizes Nrf2 protein and induces Nrf2-target genes via an antioxidant-/electrophile-response element. In mouse embryonic fibroblasts, the ability of H2S to protect against cell death caused by the redox-cycling agent menadione is dependent on Nrf2. Moreover, Nrf2 regulates murine genes involved in the production of H2S (Cystathionine-beta-synthase [Cbs] and Cystathionine-gamma-lyase [Cse]) and the degradation of H2S (Sulfide:quinone reductase-like [yeast] [Sqrdl]). We found that H2S stabilizes Nrf2 through inhibition of Keap1, an event that requires covalent modification of amino acids C226 and C613 in the substrate adaptor. Upregulation of Nrf2 by H2S partially involves the production of H2O2, which inhibits Keap1 by stimulating the formation of an intramolecular disulfide bond between C226 and C613. The Keap1 C226 and C613 residues are also S-sulfhydrated by H2S, and this may entail reduction of the C226-C613 disulfide bridge formed by H2O2. Innovation: Upregulation of Nrf2 by H2S and H2O2 involves inactivation of Keap1 through modification of C226 and C613. Conclusion: Inhibition of Keap1 by H2S leads to Nrf2-mediated induction of cytoprotective genes. Nrf2 controls Cbs, Cse, and Sqrdl, suggesting that a feedback loop exists between Nrf2 and H2S. Antioxid. Redox Signal. 00, 000-000.