INTRODUCTION: Ulcerative colitis (UC) is a major inflammatory condition worldwide. OBJECTIVES: The purpose of this study was to investigate the potential contribution of Lactobacillus johnsonii against UC from the perspective of gut microbiota-macrophage-host interactions. METHODS: L. johnsonii abundance in UC patients and colitis mice was evaluated by genomic sequencing. SPF and macrophage-depleted mice were employed to explore the effects of L. johnsonii and its products on colitis. An in vitro macrophage and intestinal epithelial cell co-culture system was constructed. Proteins in extracellular vesicles (EVs) were identified by proteomic analyses, and host signaling pathways were analyzed with transcriptomic analyses. RESULTS: L. johnsonii abundance was found to be associated with macrophage polarization and intestinal barrier function in human UC patients and mice of a colitis model. L. johnsonii and its derived EVs alleviate colitis in mice in a macrophage-dependent manner. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein in EVs derived from L. johnsonii, counteracts colitis in vitro and in vivo by regulating macrophage phenotype. GAPDH enhances anti-inflammatory macrophage polarization by inhibiting the MAPK-STAT3 axis. Macrophage-secreted EVs enhances intestinal barrier function in colitis mice by blocking the TLR4 pathway. Protein components in macrophage-derived EVs contribute to colitis remission and intestinal barrier protection. CONCLUSION: GAPDH originating in L. johnsonii-derived EVs alleviates colitis and improves intestinal barrier by inhibiting STAT3 in macrophages. EVs created from L. johnsonii are a potential novel treatment strategy for UC.