Hydrogen peroxide (H₂O₂) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H₂O₂tolerance remain unclear. Here we used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genome, to identify genes implicated in H₂O₂tolerance in Escherichia coli. Our TraDIS analysis identified 10 mutants with fitness defect upon H₂O₂exposure, among which previously H₂O₂-associated genes (oxyR, dps, dksA, rpoS, hfq and polA) and other genes with no known association with H₂O₂tolerance in E. coli (corA, rbsR, nhaA and gpmA). This is the first description of the impact of gpmA, a gene involved in glycolysis, on the susceptibility of E. coli to H₂O₂. Indeed, confirmatory experiments showed that the deletion of gpmA led to a specific hypersensitivity to H₂O₂comparable to the deletion of the major H₂O₂scavenger gene katG. This hypersensitivity was not due to an alteration of catalase function and was independent of the carbon source or the presence of oxygen. Transcription of gpmA was upregulated under H₂O₂exposure, highlighting its role under oxidative stress. In summary, our TraDIS approach identified gpmA as a member of the oxidative stress defense mechanism in E. coli.