Hydrogen peroxide (H₂O₂) is a key defense component of host-microbe interaction. However, H₂O₂concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H₂O₂concentration on gene expression of E. coliBW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H₂O₂. H₂O₂exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor OxyR leading to up-regulation of antioxidant systems, including the catalase-encoding gene (katG), that rapidly degrade extracellular H₂O₂and promote bacterial survival. We documented a strong and transient up-regulation of genes involved in sulfur metabolism and cysteine biosynthesis, which are under the control of the transcription factor CysB. This strong specific transcriptional response to H₂O₂exposure had no apparent impact on bacterial survival, but possibly replenishes the stores of oxidized cysteine and glutathione. In summary, our results demonstrate that different stress response mechanisms are activated by H₂O₂exposure and highlight the cysteine synthesis as an antioxidant response in E. coli.