Baicalin is reported as an effective drug for ulcerative colitis (UC). However, its effect on gut microbiota and short-chain fatty acids (SCFAs) remains unknown. In this study, we investigated the role of baicalin on Th17/Treg balance, gut microbiota community, and SCFAs levels in trinitrobenzene sulphonic acid (TNBS)-induced UC rat model. We found the DAI scores were significantly increased in the TNBS-treated rats, while reduced in the baicalin-treated group in a dose-dependent manner, accompanied with the alleviation of mucosal injury, the reduction of ZO-1, Occludin, and MUC2 expression. At the meanwhile, baicalin repressed the increased levels of reactive oxygen species (ROS) and MDA, while deceased the GSH and SOD levels in colon tissue of rats treated with TNBS. On the other hand, administration of baicalin attenuated the TNBS-induced upregulations of Th17/Treg ratio, indicating a strong amelioration in the colorectal inflammation. More importantly, pyrosequencing of the V4 regions of 16S rRNA genes in rat feces revealed a deviation of the gut microbiota in response to baicalin treatment. In particular, the decreased Firmicutes-to-Bacteroidetes ratios and endotoxin-bearing Proteobacteria levels indicated that baicalin reversed TNBS-induced gut dysbiosis OTUs. In addition, we further investigated the fecal levels of major SCFAs in rats and found that baicalin significantly resorted the fecal butyrate levels in rats treated with TNBS. The increased butyrate levels were in consistent with the higher abundance of butyrate-producing species such as Butyricimonas spp., Roseburia spp., Subdoligranulum spp., and Eubacteriu spp. in baicalin-treated group. In conclusion, our findings suggest that baicalin possibly protected rats against ulcerative colitis by regulation of Th17/Treg balance, and modulation of both gut microbiota and SCFAs. Baicalin may be used as a prebiotic agent to treat ulcerative colitis-associated inflammation and gut dysbiosis.