Objective To establish a non-alcoholic fatty liver disease (NAFLD) model in Wistar-SD Hypercholesterolemia (WSHc) rats induced by a high-fat diet and to reveal the pathogenesis of NAFLD in these rats through the Srebp-1 gene. Methods After 2 weeks of dietary treatment, thirty 6-week-old WSHc rats were divided into control group, AAV empty vector group, and AAV group, with 10 rats in each group. The vector group and AAV group were intravenously injected with vector virus and shRNA containing virus respectively. WSHc rats were fed with normal fat diet as a control group. Serum levels of ALT, AST, TBIL, ALP, TBA, GLU, CHOL, and TG were measured every two weeks. After further 8 weeks of feeding, the rats were euthanized and livers were excised for HE staining, Oil Red O staining, Masson staining, and Sirius red staining to observe the morphology, lipid deposition, and fibrosis of the liver tissues. RT-PCR was performed to detect the expression of lipid metabolism-related genes such as Srebp-1, Aacs, FASN, and LDLR in the livers. Furthermore, hepatocytes were isolated, cultured and divided into blank control group and high-fat control group. Then, expression of the Srebp-1 gene was detected by RT-PCR. Srebp-1 knockout (KO) hepatocytes was constructed, afterward, TG content was detected and the lipid accumulation was observed by Oil Red O staining. Results After 10 weeks of high-fat diet treatment, serum ALT (P<0.001), ALP (P<0.001), TBA (P<0.05), GLU (P<0.001), and CHOL (P<0.001) significantly increased in WSHc rats. Abnormal lipid deposition with formation of large vacuolar lipid droplets and fibrotic lesions in livers were observed. The mRNA expression of Srebp-1 obviously increased in WSHc rats (P<0.001). Moreover, compared with the vector group, the ALT (P<0.05) and GLU (P<0.01) in the WHSc+AAV group decreased, alleviated liver lipid deposition and formation of large vacuolar lipid droplets. Expressions of genes such as FASN (P<0.05) and LDLR (P<0.01) were significantly upregulated. Additionally, there was a significant increase in the expression of Srebp-1 in hepatocytes of the high-fat control group (P<0.001), while after Srebp-1 gene knockout, cellular TG levels decreased and the degree of lipid droplet aggregation was reduced. Conclusion The Srebp-1 gene plays a regulatory role in hepatic lipid metabolism and deposition, modulating the expression of lipid metabolism-related genes in WSHc rats with NAFLD. In vitro experiments demonstrated that downregulation of Srebp-1 alleviates lipotoxic injury in hepatocytes, suggesting that the development of NAFLD in WSHc rats is closely associated with abnormally high expression of the Srebp-1 gene.