In recent years, an increasing number ofListeria monocytogenesstrains with resistance to quaternary ammonium compounds (QACs) have been reported. However, the genetic basis for QACs resistance inL. monocytogenesremains poorly understood. In the present study, we have characterized the operonlmo0852/lmo0853/lmo0854(designatedsugR/sugE1/sugE2) that contributes to QACs' resistance inL. monocytogenesEGD-e. We constructed the gene deletion mutants and the complemented strains, determined minimum inhibitory concentrations (MICs) of these strains against antimicrobial agents, assessed the transcription levels of target genes by RT-qPCR, and measured the promoter activity by using beta-galactosidase assays. We also investigated the interaction between the promoter DNA and the putative regulatory protein by electrophoretic mobility shift assay (EMSA). Thesugoperon consists of a putative TetR family regulator encoded bysugRand two small multidrug resistance (SMR) efflux pumps encoded bysugE1andsugE2. Our results showed that either SugE1 or SugE2 is sufficient for QACs' resistance, indicating their function overlapping in QACs' resistance. Interestingly, lacking onesugEgene could lead to a significant increase in transcription of the othersugEgene in the presence of benzalkonium chloride (BC). Additionally, SugR negatively regulates the transcription of thesuglocus by binding to the operon promoter. Given that QACs are commonly used in food industry, the findings from this study will help us to have a better understanding of the adaptations ofL. monocytogenesto this type of disinfectant.