Although estrogen is known to play a role in fatty acid metabolism, it remains unclear whether fatty acid oxidation in mature female rats differs from fatty acid oxidation in peri-pubertal young rats. In this study, we measured fatty acid metabolism in the skeletal muscles and livers of 5 and 50 weeks old male and female rats. The rate of palmitate oxidation in the liver and gastrocnemius red in the 50-week-old female rats were elevated as compared to the 5-week-old females, whereas there were no differences in the male rats. The rate of palmitate oxidation in the gastrocnemius red was correlated inversely with intra-abdominal fat mass in the 5-week-old male and female rats, whereas the palmitate oxidation rate was positively correlated with fat mass in the liver and gastrocnemius red in the 50-week-old rats. HOMA-IR and plasma insulin levels were positively correlated with intra-abdominal fat mass in the pooled 50-week-old male and female rats, but this correlation was not apparent in 5-week-old rats. In summary, the rate of fatty acid oxidation measured in the middle-aged adult female rats was significantly higher than those measured in the peri-pubertal young female rats. This difference may be attributed to the influence of ovarian hormones.