[Background] Quaternary ammonium compounds, especially benzalkonium chloride (BC), are widely used in food industry, leading to decreased sensitivity to BC in Listeria monocytogene. Efflux pumps are recognized as an important mechanism for BC tolerance. [Objective] To investigate the role of efflux pump MdrL in tolerance to BC in Listeria monocytogenes. [Methods] The mutant strain ΔmdrL was constructed from the wild-type strain EGD-e by homologous recombination. The differences in BC tolerance, growth in sub-lethal concentration of BC (2 μg/mL), and survival in lethal concentration of BC (16 μg/mL) between the mutant strain ΔmdrL and the wild-type strain EGD-e were investigated. [Results] The mutant strain ΔmdrL and its complemented strain were constructed in this study. The mutant and wild-type strains showed the same minimum inhibitory concentrations to BC. The results show that compared with the wild-type strain EGD-e, the lag-phase duration of the mutant strain ΔmdrL was prolonged and the mean maximum growth rate and the mean maximum optical density were decrease in the presence of sub-lethal concentration of BC. Significantly difference was observed in the serial diluted culture between the mutant strain ΔmdrL and the wild-type strain EGD-e on the plates in the presence of BC (4 μg/mL). Compared with the wild-type strain EGD-e, two log decrease were observed in the mutant strain ΔmdrL in the presence of the lethal concentration of BC. The results of electron microscopy showed that the mutant strain ΔmdrL became longer when exposed to BC. Besides, no differences were observed in the accumulation and efflux to ethidium bromide (EB) between the mutant strain ΔmdrL and the wild-type strain EGD-e. [Conclusion] Efflux pump MdrL plays an important role in tolerance to BC in Listeria monocytogenes, however, this efflux pump is not associated with efflux of EB.