Iron cycling mediated by iron-reducing bacteria is an important factor driving material cycle in the surface system of earth. H₂O₂ naturally generated and artificially injected into the subsurface environment may affect the activity and function of iron-reducing bacteria through oxidative stress, but the response and mechanism of iron-reducing bacteria to H₂O₂ disturbance is still unclear. In this study, Shewanella oneidensis MR-1 was chosen as a representative iron-reducing bacterium. In combination with batch experiments and RNA-seq analysis, the changes of MR-1 activity and function under different concentrations of H₂O₂ and its regulatory mechanism were investigated. Results show that MR-1 could resist H₂O₂ stress effectively, and H₂O₂ enhanced the Fe(III)-reducing ability of MR-1. RNA-seq results show that MR-1 maintained in an anti-stress state infacing to H₂O₂ disturbance, which could resist the negative effects of H₂O₂ by actively oxidizing organic matter to provide energy and promoting the synthesis of catalase.