Abstract: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease closely associated with metabolic syndrome, and its animal models are essential for investigating the pathological mechanisms and disease progression of NAFLD. Currently, the most commonly used NAFLD animal models include the high-fat, methionine- and choline-deficient diet (HFMCD) and the high-fat diet (HFD)-induced mouse models. In this study, we compared NAFLD mouse models established by an 8-week HFMCD diet and a 12-week HFD, systematically evaluating differences in liver weight, histological alterations, lipid metabolism, and inflammation-related gene expression. The experimental results showed that, compared with the normal chow diet (NCD) group, mice in the HFMCD group exhibited significant reductions in body weight and blood glucose levels, whereas those in the HFD group exhibited significant increases. Both dietary models led to an increase in liver weight; however, hematoxylin and eosin (H&E) staining revealed more severe liver damage in the HFMCD group. Oil red O staining indicated a significantly greater degree of hepatic lipid accumulation in the HFMCD group than in the HFD group. Quantitative PCR (qPCR) analysis demonstrated that, compared with the NCD group, both the HFMCD and HFD groups exhibited significantly elevated hepatic expression of inflammatory cytokines. Sirius red staining demonstrated pronounced hepatic fibrosis in the HFMCD group, accompanied by a significant upregulation of genes associated with collagen synthesis, whereas the HFD group exhibited no evident fibrotic alterations. F4/80 immunohistochemical staining showed increased hepatic macrophage infiltration in both models, with a significantly higher number of F4/80-positive macrophages in the HFMCD group than in the HFD group. In conclusion, the HFMCD and HFD-induced NAFLD models exhibit distinct pathological characteristics and molecular mechanisms. The HFMCD model is more suitable for studying the progression of NAFLD to non-alcoholic steatohepatitis (NASH), whereas the HFD model is more appropriate for investigating the metabolic mechanisms of simple steatosis. This study provides experimental evidence for NAFLD research and offers guidance for selecting appropriate animal models based on specific research objectives. All experimental animals used in this study were approved by the Animal Care and Use Committee of Nanjing University and were handled in strict accordance with the guidelines and recommendations provided by the Nanjing University's Animal Ethics Committee.