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Context: Chinese herb Huangqin decoction (HQD) can regulate intestinal flora in ulcerative colitis (UC) mice., Objective: Our study clarifies the mechanism of HQD in regulating the intestinal flora of UC mice., Materials and Methods: Male C57BL/6 mice were randomly divided into six groups: Control, Model (3% DSS), Sulfasalazine (500 mg/kg), HQD-L (250 mg/kg), HQD-M (500 mg/kg), and HQD-H (1000 mg/kg) groups. Measurement of body weight, colon length, DAI, and haematoxylin-eosin staining were conducted. FISH and 16S rDNA detected colonic bacterial infiltration and intestinal flora changes. The expression of RegIIIγ and PRRs (NOD2, TLR5, TLR4) were detected by FCM and WB, respectively. In addition, WB, qPCR, or IHC were used to detect the expression of NOD2, MyD88, RIP2, and NF-κB p65 in the colon. ELISA was used to determine cytokines., Results: Compared with the model group (DAI score, 2.38 ± 0.05; histological score, 4.08 ± 0.54), HQD treatment significantly reduced the DAI score (L, 2.16 ± 0.09; M, 1.45 ± 0.05; H, 1.18 ± 0.05) and histological score (L, 3.16 ± 0.82; M, 2.50 ± 0.81; H, 1.51 ± 0.76); restored the weight, the colonic length ( p  < 0.05). 16S rDNA identification showed HQD regulated the balance of intestinal flora. Moreover, HQD suppressed the expression of RegIIIγ ( p  < 0.05) and prevented colonic bacterial infiltration. Furthermore, WB results showed NOD2, and TLR4 were inhibited by HQD, especially NOD2 ( p  < 0.01). The data of WB, qPCR, and IHC demonstrated that the NOD2-dependent pathway was inhibited by HQD ( p  < 0.01)., Discussion and Conclusions: HQD (1000 mg/kg) regulates the intestinal flora of colitis mice, mainly characterized as inhibition of the NOD2-dependent pathway. These results indicate that HQD has potential.