Reuterin is a broad-spectrum antimicrobial small molecule produced from glycerol by through a one-step enzymatic reaction. In this study, we conducted a systematic analysis of the Keio single-gene knockout mutant library to elucidate the molecular and cellular mechanisms of reuterin. Using sublethal concentrations of reuterin, we identified 159 sensitive mutants and 117 resistant mutants. GO and KEGG pathway analyses revealed that gene deletions in pathways related to the synthesis of aromatic amino acids, sulfur metabolism, and glutathione metabolism result in metabolic vulnerabilities that contribute to the antimicrobial mechanism of reuterin. This suggests that reuterin disrupts the intracellular redox balance, leading to reactive oxygen species production that the cells cannot manage, ultimately inducing cell death. Furthermore, the deletion of biofilm-related genes ( and ) was found to increase biofilm formation, which functions as a structural barrier, reducing the uptake of environmental drugs into the cells and contributing to resistance against reuterin. Protein-protein interaction network analysis identified a series of antioxidant defense-related proteins that form the functional network underlying 's resistance to reuterin. Taken together, these findings enhance our understanding of the antibacterial activity mechanism of reuterin and provide potential targets and theoretical support for the development of new antimicrobial agents and further study of bacterial resistance mechanisms.IMPORTANCEReuterin is a low-molecular-weight compound with broad-spectrum antimicrobial activity. A systematic analysis was conducted using a library of non-essential gene deletion strains of to elucidate the overall picture of its mechanism of action. While the generation of reactive oxygen species was suggested, this study was able to clarify the reactions occurring within cells that took up reuterin. It was revealed that pathways related to the synthesis of aromatic amino acids, sulfur metabolism, and glutathione metabolism play crucial roles in the antimicrobial mechanism. These findings not only deepen the understanding of the mechanism behind reuterin's antimicrobial activity but also provide important information for considering the physiological significance of the antimicrobial activity of molecules secreted by other microorganisms in the intestinal environment.