Objective To investigate the effect of Changyu Enema Prescription in the treatment of ulcerative colitis (UC) and related mechanism of action. Methods A total of 64 specific pathogen-free rats were randomly divided into normal control group, model group, mesalazine group, and Changyu Enema Prescription group, using a random number table. Colon perfusion with 2,4,6-trinitrobenzenesulfonic acid was performed to establish a rat model of UC, and disease activity index (DAI) was observed on day 12 of the experiment. After the experiment ended, ELISA was used to measure the levels of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the colonic mucosa. Results Compared with the model group, the Changyu Enema Prescription group and the mesalazine group had a significant reduction in DAI, a significant increase in serum TGF-β1, and a significant reduction in TNF-α (all P<0.05). Conclusion Changyu Enema Prescription has a marked effect in the treatment of UC, possibly by reducing the serum levels of proinflammatory factors and promoting the expression of anti-inflammatory cytokines.