As a pathogenic microorganism, Listeria monocytogenes is widely used in the study of bacterial pathogenic mechanism and host defense mechanism. The lysosomal escape of Listeria monocytogenes is essential for its replication in the cytoplasm. Here, we report that Listeria monocytogenes infection can cause the expression level of Six-transmembrane epithelial antigen of the prostate 3 (Steap3) to decrease in macrophages. Functional experiments show that Steap3 can inhibit lysosomal escape of Listeria monocytogenes. Then, the comprehensive proteomic analysis reveals that the deletion of Steap3 affects the lysosomal signaling pathway of macrophages. Among these proteins affected by Steap3, we discover that Ganglioside GM2 activator (Gm2a) may be a potential target gene of Steap3. In summary, we find that Steap3-Gm2a axis can inhibits lysosomal escape of Listeria monocytogenes and serve the potential molecular drug targets for antibacterial treatment.