This work was undertaken to study the regulatory effects of the Agr and LuxS/AI-2 quorum sensing systems on biofilm formation by Listeria monocytogenes (Lm). The agrD and luxS genes of LMB33426 were knocked out by homologous recombination, and the biofilm formation characteristics of the wild-type and mutant strains were investigated comparatively. The results showed that the biofilm formation ability of ΔagrD and ΔluxS was reduced compared with that of the wild-type strain. The hydrophobicity of ΔagrD decreased significantly, and its swimming mobility was higher than that of the wild-type strain at 37 ℃. However, no significant difference was found in drug resistance between the wild-type and mutant strains. This study provides a basis for further research on the regulatory mechanism of quorum sensing systems on biofilm formation by Lm and more broadly, for the development of novel strategies for the prevention and control of Lm infections.