Objective To explore the therapeutic effects and potential activity mechanisms of intestinal flora on a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced intestinal fibrosis rat model. Methods 24 SD rats were randomly divided into normal control, model, lincomycin hydrochloride (85 mg/ kg), and probiotic (850 mg/ kg) groups. Except for the normal control and model groups, which were given equal volumes of normal saline, the groups were given corresponding drugs by gavage, once a day, for five consecutive days. The next day, TNBS was used to induce the rat intestinal fibrosis model in all groups except for the normal control group. The corresponding drugs were then given for 7 d. During the experiment, the general behavior of the rats was observed. After the experiment, colonic specimens were collected for histological scoring. HE and Masson staining were used to observe the degree of colon tissue damage and fibrosis. The immunohistochemical detection of E-cadherin, α-SMA, TGF-β1, and other proteins and the Western Blot detection of TL1A and DR3 proteins were carried out. Results Compared with the normal control group, the model group rats’ colons were damaged, and the number of collagen fibers increased, indicating that the intestinal fiber model was successful. Lincomycin hydrochloride further aggravated colonic injury and collagen fiber expression, and colonic injury and fibrosis were alleviated by probiotic treatment. Compared with the model group, the lincomycin hydrochloride group had increased expression of TL1A/ DR3 protein (P< 0. 05) and decreased expression of E-cadherin, α-SMA, and TGF-β1 proteins (P< 0. 05). However, the probiotics group had significantly reduced protein expression levels of TL1A/ DR3, α-SMA, and TGF-β1, and increased levels of E-cadherin. Conclusions Microflora disorder promotes fibrosis by activating TL1A/ DR3 signaling to regulate epithelial mesenchymal transformation in colon tissue, and probiotic intervention can alleviate colonic fibrosis.