Background: Disturbance of the gut microbiota may play a critical role in the progression of nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD). Changes in gut microbiota were analyzed in a rat HFD-induced NAFLD model following treatment with Qinghua Fang (QHF), a Chinese herbal formula currently used in the clinical practice of traditional Chinese medicine. Methods: Sixty Wistar rats were randomly divided into 6 groups (n=10): blank group [normal chow (NC) group], model group (HFD group), control group (BG group), Qinghua Fang high-dose group [QHF(H) group], QHF mid-dose group [QHF(M) group], QHF low-dose group [QHF(L) group]. The high, medium and low doses of QHF were used to intervene in the H, M, and L groups; the BG group was given berberine; the NC and HFD groups were given distilled water for 10 weeks. H&E staining, determination of serum liver function and blood lipid levels, and changes in the structure of rat intestinal flora through 16S rDNA denaturing gradient gel electrophoresis sequencing technology and ERIC-PCR fingerprinting were performed.Results: The liver function and blood lipid levels of the rats in the HFD group were higher than those in the NC group; the alanine aminotransferase levels in the QHF-H group, QFH-M group and QHF-L group were lower than in the HFD group (P<0.05); the liver pathology of the QHF-M group and QHF-H group showed a small amount of fatty cell infiltration, but was significantly less than the hepatocyte inflammation and necrosis in the HFD group. The ERIC-PCR fingerprint and diversity analysis found that the composition of the intestinal flora of rats in the QHF-H group was significantly different from that of the NC and HFD groups. The flora of the QHF and NC groups was more diverse and richer than in the HFD group (P<0.05).Conclusions: QHF alleviated the liver dysfunction and increased blood lipid levels of NAFLD rats induced by HFD. It also effectively reduced the degree of liver steatosis and adjusted the number and structure of intestinal flora. Treatment with QHF had a significant effect on NAFLD.