【Objective】 To generate Listeria monocytogenes strains with lmo0880 deleted and complemented strains, so as to investigate the roles of Lmo0880 in bacterial infection in a host. 【Methods】 The lmo0880-deleted strain was generated by homologous recombination, and the complementary strain was constructed by introducing an integrative plasmid carrying lmo0880 into the lmo0880-deleted strain. The growth, adhesion, invasion, and intracellular proliferation were compared between the lmo0880-deleted strain, complementary strain, and the wild type. 【Results】 The deletion of lmo0880 did not significantly impact bacterial growth or adhesion. However, it led to notable decreases in cell invasion, proliferation, and colonization in the liver and spleen, ultimately diminishing the pathogenicity in mice. 【Conclusion】 The LPXTG-anchored protein Lmo0880 plays a crucial role in bacterial invasion, proliferation, and colonization in a host. These findings provide a solid foundation for deeply understanding the pathogen-host interaction during L. monocytogenes infection.