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Listeria monocytogenes is a foodborne bacterial pathogen that seriously threatens dairy product safety and human health, hence effective and available antibacterial agents are urgently needed. This study aims to investigate the antibacterial mechanism of a recombinant enterocin CHQS against L. monocytogenes and its antibacterial effect on pasteurized milk. The minimum inhibitory concentration (MIC) of CHQS was 0.5 μg/mL in both TSB medium and tenfold diluted pasteurized milk. The anti- L. monocytogenes rate of 2 h reached 99.94% with the treatment of the 2 MIC of CHQS in the diluted pasteurized milk. CHQS led to the massive release of proteins and nucleic acids and the significant increase in propidium iodide (PI) uptake, indicating that integrity and permeability of the cell membrane were destroyed. The cell membrane and structure of L. monocytogenes were severely disrupted by observing with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) level was increased to 2.18-fold with the 2 MIC CHQS treatment. CHQS at sub-MICs inhibited the biofilm formation and motility of L. monocytogenes in a concentration-dependent manner. In MIC treatment group, the number of L. monocytogenes was 4.55 log 10 CFU/mL fewer than the control group and the bacterial abundance of L. monocytogenes remained a low level (0.22%) on the 12th day of storage at 4 °C in pasteurized milk. The study suggested the antibacterial mechanism of CHQS and its application potentiality in pasteurized milk. • The antibacterial mechanism of a recombinant enterocin CHQS against L. monocytogenes was investigated. • The antibacterial effect of CHQS on pasteurized milk was explored. • CHQS inhibited L. monocytogenes growth by disrupting the integrity and permeability of cell membranes. • CHQS can stimulate ROS generation and inhibit the motility and biofilm formation of L. monocytogenes. [ABSTRACT FROM AUTHOR]