The integrity of gut barrier functions is closely associated with the pathogenesis of colitis. It is speculated that Lactobacillus brevis Bmb6 alleviates colitis by improving the tight junction (TJ) of the inflamed intestinal epithelial layer. In the present study, the regulatory effects of L. brevis Bmb6 on the TJ barrier to ameliorate colitis-symptoms were investigated. Preliminary screening showed that L. brevis Bmb6 exhibited strong acid and bile acid tolerance, along with antioxidants and β-galactosidase activities. In a 14-day dextran sulfate sodium (DSS)-induced colitis mouse model, treatment with L. brevis Bmb6 significantly decreased in the disease activity index score. In addition, histological analyses showed that treatment with L. brevis Bmb6 protected the structural integrity of the intestinal epithelial layer and mucin-secreting goblet cells from DSS-induced damage, with only slight infiltration of immune cells. Interestingly, western blotting analyses showed that the expression of the TJ protein, zona occluden-1, was restored in Bmb6-treated mice, but not in DSS-induced mice. Consistently, the gene expression of inflammatory cytokines (tumor necrosis factor-α and interferon-γ) was also suppressed in the Bmb6-treated mice. Hence, our findings suggest that suppression of inflammatory conditions enhanced expression of TJ protein, ZO-1, or vice versa, contributing to a colitis-ameliorating effect in L. brevis Bmb6.