Objective This study sought 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 High-fat control group, HFD + AAV no load group, and HFD + AAV group, with 10 rats in each group. The HFD + AAV no load group and HFD +AAV group were intravenously injected with a vector virus and an shRNA containing virus, respectively. WSHc rats were fed with a normal fat diet as a normal control group. Serum levels of ALT, AST, TBIL, ALP, TBA, GLU, CHOL, and TG were measured every 2 weeks. After a 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-qPCR was performed to detect the expression of lipid metabolism-related genes namely Srebp-1, Aacs, FASN and LDLR in the livers. Furthermore, hepatocytes were isolated, cultured, and divided into a normal control group and a high-fat control group. Next, expression of the Srebp-1 gene was detected by RT-qPCR. Srebp-1 knockout (KO)hepatocytes were constructed, then 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 noticeably increased in WSHc rats (P<0.001). Moreover, compared with the high-fat control group, the ALT (P<0.05)and GLU (P<0.01)in the HFD + AAV group decreased, and liver lipid deposition and the formation of large vacuolar lipid droplets were alleviated. 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. Conclusions 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 expressions of the Srebp-1 gene.