Abstract Recent studies have shown that intestinal commensal bacteria-derived vesicles may have potential effects in alleviating ulcerative colitis (UC). Although Bifidobacterium longum is widely used to prevent colitis, the potential role of B. longum-derived extracellular vesicles has yet to be explored. Here, we extracted B. longum NSP001-derived extracellular vesicles (NEVs) and investigated the regulatory roles of NEVs in colitis. Our results demonstrated that NEVs alleviate UC by improving intestinal barrier, modulating immune cell differentiation, and promoting the production of SCFAs. NEVs’ improvement of inflammation in pseudo-germ-free mice implies that the anti-inflammatory effect of NEVs does not exclusively depend on the regulation of gut microbiota. In conclusion, we suggest that B. longum NSP001 improves UC through the secretion of NEVs. In addition, the study emphasizes the critical role of NEVs in maintaining host immune homeostasis via suppressing STAT3 pathway, thereby highlighting their potential as a novel postbiotic to alleviate UC.