Background: An unbalanced dietary intake of omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) has been associated with chronic inflammation and oxidative stress, both of which contribute to the pathophysiology of obesity. Objective: We aimed to evaluate the effects of a diet with an n-6:n-3 PUFA ratio of 5:1 on body composition, biochemical parameters, and the gene expression of cytokines and antioxidant enzymes in a murine model of diet-induced obesity. Methods: A diet-induced obesity model was established in C57BL6/J mice over 17 weeks. Mice were then fed different diets for 8 weeks: a control diet (chow), a high-fat diet with a 30:1 n-6:n-3 ratio (HFD-30:1), and a high-fat diet enriched with n-3 fatty acids, with a 5:1 n-6:n-3 ratio (HFD-5:1). Body weight and food intake were monitored throughout this study. Biochemical parameters were measured, and the expression of antioxidant enzymes and cytokine genes was analyzed by qPCR. Data were analyzed using GraphPad Prism software. Results: The HFD-5:1 group exhibited a significant reduction in body weight (p = 0.0182), liver tissue weight (p = 0.01), serum glucose levels (p = 0.010), area under the curve (AUC) (p = 0.0161), cholesterol (p < 0.0001), and triglycerides (p = 0.0069) compared to the HFD-30:1 group. The body weight in the HFD-5:1 group decreased to levels comparable to the control group. Additionally, the expression of the inflammatory cytokine genes Ccl2 (p = 0.0389) and Tgfb1 (p = 0.0226) was significantly reduced. Conclusions: These findings suggest that adjusting the dietary n-6:n-3 ratio to 5:1 modulates inflammation-related gene expression and improves metabolic markers in obese mice, supporting its potential relevance for future translational research.