Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. It is asymptomatic at presentation and is frequently identified among individuals with metabolic dysfunction, including obesity and diabetes. NAFLD is primarily characterized by the accumulation of triacylglycerol in the liver. Since insulin resistance and fat metabolism dysregulation are major causes of type 2 diabetes and NAFLD, anti-diabetes agents are widely considered as potential therapy strategies for NAFLD. Sitagliptin, an inhibitor of dipeptidyl peptidase-4, has been developed as an oral anti-hyperglycemic agent. In the present study, the effect of sitagliptin on the progression of NAFLD was evaluated in a rat model fed with a high fat diet (HFD). It was identified that sitagliptin significantly suppressed lipid accumulation in rat blood and liver and improved insulin resistance. Furthermore, it was revealed that sitagliptin reactivated the HFD-suppressed SIRT1/AMPK axis pathway and upregulated its downstream target genes, modulating fatty acid metabolism. These findings demonstrate a preventive effect of sitagliptin on hepatic lipid dysregulation and suggest that sitagliptin has potential as a clinical therapeutic strategy for NAFLD.