Dietary supplementation with CLA has been reported to alleviate the effect of colitis in mice, but the mechanisms involved need further exploration. The study aimed to investigate how orally administered CLA alleviates DSS-induced colitis in mice. CLA was administered at five different doses: 40 mg/d, 20 mg/d, 10 mg/d, 5 mg/d and 2.5 mg/d. Doses of CLA at 10 mg/d and higher alleviated colitis symptoms and reduced inflammation induced by DSS, in which 40 mg/d, 20 mg/d and 10 mg/d CLA significantly increased the concentration of MUC2 and goblet cells, but neither 5 mg/d CLA nor 2.5 mg/d CLA had any effects. Meanwhile, 40 mg/d CLA and 20 mg/d CLA treatments significantly up-regulated the concentration of tight junction proteins (ZO-1, occludin and claudin-3) and ameliorated epithelial apoptosis caused by DSS. Moreover, oxidative stress-related enzymes (SOD, GSH-PX, CAT) and inflammatory cytokines (TNF-a, IL-10, IL-6) were modulated by 40 mg/d CLA and 20 mg/d CLA. Furthermore, 40 mg/d CLA rebalanced the gut microbiota damaged by DSS, including reducing Bacteroides and increasing Bifidobacterium and Odoribacter. In conclusion, CLA supplementation alleviated DSS-induced colitis in a dose-dependent manner by modulating inflammatory cytokines and oxidation stress, maintaining the mucosal barrier and reverting microbiota changes.