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Listeria monocytogenes (<i>L. monocytogenes</i>) is a foodborne pathogen that causes listeriosis in humans and other animals. Surface proteins with the LPXTG motif have important roles in the virulence of <i>L. monocytogenes</i>. Lmo0159 is one such protein, but little is known about its role in <i>L. monocytogenes</i> virulence, motility, and biofilm formation. Here, we constructed and characterized a deletion mutant of <i>lmo0159</i> (∆<i>lmo0159</i>). We analyzed not only the capacity of biofilm formation, motility, attachment, and intracellular growth in different cell types but also LD₅₀; bacterial load in mice’s liver, spleen, and brain; expression of virulence genes; and survival time of mice after challenge. The results showed that the cross-linking density of the biofilm of ∆<i>lmo0159</i> strain was lower than that of WT by microscopic examination. The expression of biofilm-formation and virulence genes also decreased in the biofilm state. Subsequently, the growth and motility of ∆<i>lmo0159</i> in the culture medium were enhanced. Conversely, the growth and motility of <i>L. monocytogenes</i> were attenuated by ∆<i>lmo0159</i> at both the cellular and mouse levels. At the cellular level, ∆<i>lmo0159</i> reduced plaque size; accelerated scratch healing; and attenuated the efficiency of adhesion, invasion, and intracellular proliferation in swine intestinal epithelial cells (SIEC), RAW264.7, mouse-brain microvascular endothelial cells (mBMEC), and human-brain microvascular endothelial cells (hCMEC/D3). The expression of virulence genes was also inhibited. At the mouse level, the LD₅₀ of the ∆<i>lmo0159</i> strain was 10^0.97 times higher than that of the WT strain. The bacterial load of the ∆<i>lmo0159</i> strain in the liver and spleen was lower than that of the WT strain. In a mouse model of intraperitoneal infection, the deletion of the <i>lmo0159</i> gene significantly prolonged the survival time of the mice, suggesting that the <i>lmo0159</i> deletion mutant also exhibited reduced virulence. Thus, our study identified <i>lmo0159</i> as a novel virulence factor among <i>L. monocytogenes</i> LPXTG proteins.