BACKGROUND: Lactobacillus fermentum has been shown to improve intestinal health and treat colitis; however, its precise efficacy and mechanisms in inflammatory bowel disease remain unclear. OBJECTIVES: This study aimed to evaluate whether L fermentum and its metabolites, extracellular vesicles, and other components could modulate intestinal barrier function and gut microbiota to alleviate dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: Forty-eight mice were randomly assigned to 6 groups: control, DSS, L fermentum+DSS group (LF+DSS), heat-inactivated L fermentum+DSS group (LHF+DSS), L fermentum supernatant solution+DSS group (LSF+DSS), and L fermentum extracellular vesicles+DSS group (LEV+DSS). After a 1-wk acclimation, mice were gavaged daily for 3 wk. Fresh cultures, including live (LF+DSS), heat-inactivated (LHF+DSS), supernatant (LSF+DSS), and extracellular vesicles (LEV+DSS), were prepared daily. During the final 7 d, the control group received normal water, and the other groups received 3% DSS. Data were collected daily, followed by sample collection from the mice. RESULTS: In this study, significant reductions (P < 0.05) in body weight changes, disease activity index, intestinal damage, and histology scores were observed in the treatment groups, especially LEV+DSS and LF+DSS. Additionally, compared with the DSS group, colonic mucus secretion, as well as claudin-1 and occludin expression, increased significantly (P < 0.05) in the LEV+DSS and LF+DSS groups, whereas proinflammatory cytokines IL-1beta and TNF-alpha decreased (P < 0.05) and IL-10 increased (P < 0.05) in the LEV+DSS group. L fermentum and its components significantly regulated gut microbiota alpha-diversity and beta-diversity, affecting overall composition. Linear discriminant analysis effect size analysis revealed an enrichment of beneficial bacteria including Prevotellaceae_UCG-001, Romboutsia, and Ruminococcus species in the LF+DSS group and Akkermansia, Odoribacter, and Marvinbryantia species in the LEV+DSS group. Both L fermentum and its extracellular vesicles significantly downregulated the gene expression of TNF-alpha and IL-1beta, whereas the expression of IL-10 was upregulated, thereby contributing to the alleviation of colitis symptoms. CONCLUSIONS: This study reveals that L fermentum alleviates colitis through modulation of the gut microbiota and reinforcement of the intestinal mucosal barrier, with its extracellular vesicles potentially playing a key role in this regulatory process.