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To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A.Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml−1. Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP.Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis.This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species-specific mode of action.