Mitochondrion is considered as the major source of intracellular reactive oxygen species (ROS). Hydrogen sulfide (H2S) a newer endogenous gaseous mediator, is reported to be anti-oxidant(1,2). However, the underlying mechanism remains largely elusive. P66Shc is an upstream activator of mitochondrial redox signaling (3,4). The aim of this study was to explore whether the antioxidant effect of H2S is mediated by p66Shc. We found that pretreatment with NaHS (an H2S donor, 100 μM) for 30 min inhibited H2O2 (50 μM, 2 h) induced mitochondrial ROS generation. Similarly, over-expression of CBS caused a significant inhibition on H2O2 induced mitochondrial oxidative stress. Both endogenous and exogenous H2S induced p66Shc sulfhydration and attenuated H2O2-induced phosphorylation of p66Sh. To identify the sulfhydrated cysteine residue in p66Shc, the conversed cysteine-59 was mutated to serine (C59S). It was found that C59S markedly attenuated p66Shc sulfhydration induced by NaHS and the inhibitory effect of H2S on H2O2-induced p66Shc phospholyration. These data suggest that H2S-induced sulfhydration may contribute to its inhibitory effect against p66Shc phospholyration. Moreover, C59S also attenuated the protective effect of NaHS against H2O2-induced mitochonridal ROS production and cell injury. Co-immunoprecipitation and Western blotting assays showed that NaHS treatment reduced the interaction between PKCβII and p66Shc in HEK293 cells treated with H2O2. However, this effect was eliminated in C59S, suggesting a key role of Cys-59 in PKCβII mediated p66Shc phosphorylation.We also evaluated the mitochondrial pool of p66Shc. H2O2 treatment increased the amount of p66Shc within mitochondria in normal SH-SY5Y cells. In contrast, the expressions of p66Shc were less pronounced in mitochondria isolated from cells pretreated with 100 μM NaHS for 30 min. Similar effect was also found in HEK293 cells transfected with WT-p66Shc. However, no detectable changes were observed in cells transfected with C59S mutants. This was further confirmed by immunofluorescence assay. Confocal microscopy confirmed a clear preferential colocalization between p66Shc and mitochondria upon treatment with H2O2 (50 μM) for 30 min and this effect was abolished by NaHS pretreatment. Our data suggest that H2S may prevent the translocation of p66Shc to the mitochondria and its contribution to ROS generation. In conclusion, H2S inhibits mitochondrial ROS production via the sulfhydration of Cys-59 residue, which, in turn, prevents the phosphorylation of p66Shc.We therefore revealed a novel mechanism for the anti-oxidant effect of H2S and its role in the oxidative stress related diseases.