Specific Lactobacillus reuteri is autochthonous Lactobacillus species in humans with potential application in food production as a probiotic. The difference in colonizing Peyer's patches (PP) might decide their health-promoting properties. We aimed to investigate the difference between PP- and lumen-specific L. reuteri on antimicrobial peptide expression in this study. L. reuteri strains were isolated from PP and the lumen of C57BL/6J mice, which were used to treat mice. PP-specific L. reuteri cells stimulate RegIIIγ mRNA expression of the crypt epithelial sample. PP-specific L. reuteri induces accumulation of extracellular DNA (eDNA) in the bottom of crypts. eDNA was extracted from the small-intestinal mucus, the yield of which was significantly increased after the PP-specific L. reuteri treatment. And it increased cytokine production in RAW264.7 murine macrophages. PP-specific L. reuteri significantly increased the relative abundance of Bacteroidetes-, Lactobacillus-, and Proteobacteria-derived eDNA. However, the levels of Strentrophomonas-derived eDNA decreased. These results provide a rationale for the screening of human derived L. reuteri with an immune-modulatory property.