[Objective] To explore the function of rmlB gene on antibiotic resistance, biofilm formation, and virulence in Listeria monocytogenes. [Methods] The rmlB gene was deleted through homologous recombination. Antibiotic resistancedifferences between the wild-type and the rmlB deletion mutant were compared. The biofilm formation ability of the mutant was measured using the microtiter plate assay. The transcription of major virulence genes in L. monocytogenes and hemolytic activity of the mutant were studied. [Results] Compared tothe wild-type strain, the deletion of rmlB increased susceptibility to envelope-acting antibiotics such as several cephalosporinsand bacitracin (P≤0.01) and decreased bacterial biofilm formation (P≤0.01).The transcription expression of the well-known virulencegene hly in L. monocytogenes and bacterial haemolytic activity were also significantly reduced in the rmlB deletion mutant (P≤0.01). [Conclusion] rmlB plays a crucial role in L. monocytogenes against the envelope-acting antibiotics, biofilm formation and virulence.