Abstract: This work aimed to investigate the inactivation mechanism of lauroyl arginate ethyl (LAE) against Listeria monocytogenes. The antibacterial activity of LAE against L. monocytogenes was evaluated by measuring the minimum antibacterial concentration (MIC). Then, the antibacterial mechanism of LAE against L. monocytogenes was investigated by measuring the cell morphology, cell membrane integrity, intracellular ATP level, cell membrane potential, cell surface hydrophobicity, and intracellular reactive oxygen species (ROS) level. The results showed that LAE could effectively inhibit the growth of L. monocytogenes with a MIC value of 10 μg/mL. After LAE treatment at a final concentration of 40 μg/mL for 10 min, the cell morphology of L. monocytogenes shrank obviously, the levels of extracellular nucleic acid and protein significantly (P<0.05) increased by 1.64- and 15.39-fold, respectively, as compared with the control cells, indicating a significant enhancement of membrane permeability. After LAE treatment at a final concentration of 40 μg/mL for 10 min, the cell membrane was depolarized and the cell surface hydrophobicity was significantly enhanced (P<0.05). The intracellular level of ATP decreased by 92.40% (P<0.05), while the intracellular ROS level increased by 77.27% (P<0.05) compared with the control cells. In addition, glutathione and N-acetyl-L-cysteine could significantly (P<0.05) reduce the antibacterial activity of LAE against L. monocytogenes. In summary, LAE can effectively inactivate L. monocytogenes, which may be associated with the damage of cell membrane and oxidative stress. This study provides a theoretical basis for the practical application of LAE in food preservation.