In the present study, to better comprehensively investigate the impact of Fat-1 on circulating and hepatic lipid metabolism, MAFLD, and atherosclerosis in different hamster models, we constructed an adeno-associated virus 9 (AAV9) vector to express Fat-1 in both WT and LDLR−/− hamsters, aiming to evaluate the vital role of Fat-1 in spontaneous and HFD-induced MAFLD and atherosclerosis, and then assess the feasibility of AAV9 expressing Fat-1 (AAV9-Fat-1) as a gene therapy approach with potential clinical applications to treat human metabolic diseases. This evidence concerns the gene LDLR and atherosclerosis.