Abstract: Objective: To investigate the effects of iron overload in conjunction with different types of high-fat diets on liver injury in mice. Methods: Forty-eight SPF male C57BL/6J mice were randomly divided into six groups based on their body weight (n=8 per group). The normal control group (ND) and normal diet with high iron group (NDFe) were fed with basal diet. The palm oil high-fat diet group (PHFD), palm oil high-fat diet with high iron group (PHFDFe), soybean oil high-fat diet group (SHFD), and soybean oil high-fat diet with high iron group (SHFDFe) were fed with their respective high-fat diets. Starting from the 10th week, mice in the NDFe, PHFDFe, and SHFDFe groups were administered iron dextran through intramuscular injection at a dosage of 100 mg/kg twice a week for 8 consecutive weeks. Meanwhile, the remaining groups were given saline injections of an equal dose until the 17th week. Blood and liver samples were collected after anesthesia to evaluate serum and liver biochemical indexes. Additionally, liver pathological changes were examined, and the expression of genes associated with iron and lipid metabolism was analyzed. Results: Compared with the respective high-fat groups, iron overload in conjunction with high-fat diet significantly decreased serum total cholesterol (TC), hepatic triglyceride (TG) and glutathione peroxidase (GSH-Px) levels while increased serum TG and alanine aminotransferase (ALT), liver index, liver iron and malondialdehyde (MDA) levels (P<0.05). Additionally, liver MDA content of the SHFDFe group was significantly higher than that of the PHFDFe group (P<0.05). Compared with the PHFD group, the mRNA expression levels of Divalentmetal-iontransporter-1 (DMT-1) and Ferroportin (FPN) were significantly increased in the PHFDFe group (P<0.05). FPN mRNA expression level was significantly upregulated in the SHFDFe group when compared with the NDFe, PHFDFe and SHFD groups (P<0.05). Moreover, the mRNA expression levels of acetyl-Coenzyme A carboxylase alpha 1 (ACC1) and Fatty Acid Synthase (FASN) were significantly decreased in the SHFDFe group compared to the SHFD group (P<0.05). Conclusion: The combination of iron overload and high-fat diets has the potential to worsen lipid metabolism disorder and oxidative stress. Additionally, when combined with iron overload, soybean oil may cause more severe liver injury compared to palm oil.