Background: Recent studies have suggested that Shenling Baizhu San (SLBZS), an complementary and alternative medical therapy for ulcerative colitis (UC), alleviate clinic symptoms by the improvement of biochemical criteria and restoration of the intestinal barrier function. SLBZS as a famous Chinese herbal formula has been reportedly used to treat UC, of which mechanism is unknown. This study investigated the therapeutic effects of SLBZS on restoring the gut microbiota in a UC rat model. Methods: We proposed a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC rat model to monitor the structural modulation of the gut microbiota. The test period was 10 days (observation for two days after modeling, treatment for 8 days by SLBZS). In this study, the level of inflammatory cytokines and activity of antioxidant enzymes in serum were ascertained by enzyme-linked immunosorbent assay (ELISA) and histological changes of colon were observed. Feces were collected for high-throughput sequencing of 16S rRNA gene. Results: SLBZS partly reduced the diversity of the gut microbiota, while the abundance of that is increased. Furthermore, at the genus level, the relative abundance of short chain fatty acids (SCFA) producing bacteria including Prevotella and Oscillospira increased, while the relative abundance of harmful bacteria including Desulfovibrio, and Bilophila decreased. Additionally, SLBZS could improve the lesions of colon and significantly reduce the expression of Interleukin-6 (IL-6) and Myeloperoxidase (MPO) and increase the activities of Superoxide dismutase (SOD) and Catalase (CAT) in rats serum. Conclusions: These results demonstrate that SLBZS may treat UC effectively by inhibiting inflammation, enhancing antioxidant capacity and regulating gut microbiota.