The probiotic bacterial strain Lactobacillus plantarum ZS2058 has been proven to alleviate colitis due to its ability to synthesize conjugated fatty acids (CFAs). To investigate the specific functions of CFAs produced by this probiotic bacterium, a-linolenic acid was isomerized by Lactobacillus plantarum ZS2058, and two different conjugated a-linolenic acid (CLNA) isomers were successfully isolated: c9, t11, c15-CLNA (CLNA1) and c9, t11, t15-CLNA (CLNA2). The effects and mechanism of CLNA crude extract and individual isomers on colitis were explored. CLNA significantly inhibited weight loss, the disease activity index, colon shortening. Additionally, CLNA improved histological damage, protected colonic mucous layer integrity and significantly upregulated the concentration of tight junction proteins (ZO-1, occludin, and claudin-3). CLNA significantly attenuated the expression of proinflammatory cytokines (TNF-a, IL-1▀, and IL-6) while upregulating the expression of the colonic anti-inflammatory cytokine IL-10 and nuclear receptor PPAR┐. Moreover, the activity of oxidative stress-related enzymes (superoxide dismutase, glutathione peroxidase and catalase) was increased by CLNA. The myeloperoxidase activity was significantly decreased by CLNA. Meanwhile, the concentrations of CLNA in the liver and colonic conjugated linoleic acid (CLA) were significantly increased in response to CLNA supplementation. Furthermore, CLNA could rebalance the gut microbial composition damaged by DSS, including increasing the a-diversity. CLNA1 and CLNA2 increased the abundance of Ruminococcus and Prevotella, respectively.