Introduction: Fructose supplementation is recognized as a risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), but its role in the progression to metabolic dysfunction-associated steatohepatitis (MASH) remains less understood. This study aimed to evaluate the effect of dietary fructose on hepatic lipid metabolism (SREBP-1c, FAS, CD36, PPAR-α, ACOX-1, and CAT-1) in a murine model of hepatic steatosis progression from MASLD to MASH, as characterized by a NAS score. Materials and Methods: Male C57BL/6 mice were fed for 20 weeks with one of the following diets: 1) control diet (CD; 10% fat, 20% protein, 70% carbohydrates); 2) CD+F (30% fructose w/v); 3) high-fat diet (HFD; 60% fat, 20% protein, 20% carbohydrates); and 4) HFD+F (30% fructose w/v). General parameters, liver histology, and expression (qPCR, immunohistochemistry) of lipid metabolism markers (SREBP-1c, FAS, CD36, PPAR-α, ACOX1, CAT-1) were evaluated. Results: Mice fed an HFD+F showed increased SREBP-1c nuclear translocation and mRNA expression of FAS and CD36, while mitochondrial β-oxidation of free fatty acids was impaired due to the inhibition of PPAR-α activity and ACOX1 mRNA levels. Conclusion: Fructose supplementation exerted a strong pro-lipogenic and antilipolytic effect on the liver of mice fed an HFD, leading to increased lipid accumulation, elevated activity, and expression of FAS and CD36, as well as impairment in mitochondrial β-oxidation of free fatty acids compared to CD-fed mice. These findings suggest that fructose-induced alterations in hepatic lipid metabolism may play a critical role in the progression from MASLD to MASH, and its impact may be influenced by the metabolic state.