Abstract: This study was designed to investigate the effect of Huangqin Tang (HQT) on gut microbiota of ulcerative colitis (UC) rats, and to explore the relationship between Huangqin Tang and ulcerative colitis and gut microbiota. Fifteen male Wistar rats were randomly divided into control group, trinitrobenzene sulfonic acid (TNBS) group and TNBS + HQT group. The model of UC rats with cell immunoreactivity was made established using the compound method (TNBS and ethanol). After 10 days of administration, 15 fecal samples were collected and total DNA was extracted from the samples to get total DNA. The primers were designed on bacterial 16S rRNA V3-V4 region sequences and Illumina Miseq platform was used for high-throughput sequencing. It was found that the principal component analysis (PCA), the principal co-ordinates analysis (PCoA) and the non-metric multidimensional scale analysis (NMDS) based on the Beta diversity distance showed that there were significant differences in the composition of the gut microbiota among the three groups (P < 0.05). Compared with the control group, the Lactobacillus of the TNBS group was significantly decreased (P < 0.05), and the Lachnospiraceae, Desulfovibrio, Roseburia, Ruminococcaceae were significantly increased (P < 0.05). Compared with the TNBS group, the Lactobacillus in the TNBS + HQT group was significantly increased (P < 0.01), and the Alistipes was significantly decreased (P < 0.05). The study suggests that the Huangqin Tang plays a role in the treatment of ulcerative colitis partially through regulating the structure of the gut microbiota.