This study evaluated the effect of fish oil rich in n-3 fatty acids on bone characteristics in Sprague-Dawley rats. Weanling male rats were randomized to receive either a diet containing high fish oil (FO), fish oil blended with corn oil (FICO), or soy oil rich in n-6 fatty acids (SO) for 4 weeks. All diets provided 70 g/kg fat based on the AIN-93G diet. Growth and biomarkers of bone metabolism were analyzed, and femur bone characteristics were measured by dual-energy X-ray absorptiometry. After the dietary treatment, no significant differences among the diet groups were observed for serum concentrations of Ca, parathyroid hormone, calcitonin, or osteocalcin. Alkaline phosphatase activity was significantly greater in FO-fed rats compared to that in the FICO and SO groups, whereas no difference in deoxypyridinoline values was observed, supporting the positive effect of a FO diet on bone formation. These results were accompanied by a significant increase in femur bone mineral density (BMD) in FO-fed rats. These findings suggest that providing fish oil rich in n-3 fatty acids correlates with higher alkaline phosphatase activity and BMD values, favoring bone formation in growing rats.