The objective of this study was to investigate the sex-dependent regulation of skeletal muscle UCP3 mRNA expression in response to overweight and its relationship with serum levels of free fatty acids, leptin and insulin. Two obesity models were used: rats made obese by feeding them with a cafeteria diet for 14 weeks, and post-cafeteria overweight rats fed a chow diet for 10 weeks after consuming the cafeteria diet for 14 weeks. The effects of 24-hour fasting were studied in post-cafeteria rats and their age-matched controls. The cafeteria rats ate a high fat diet and attained an excess body weight that was higher in females (+59%) than in males (+39%). A trend to higher induction of abdominal muscle UCP3 mRNA in male rats than in females after cafeteria diet was apparent (+116% increase versus +26% increase). Post-cafeteria male, but not female rats, still showed the tendency to have increased UCP3 mRNA levels relative to their age-matched controls. A linear regression analysis showed a significant positive correlation of the UCP3 mRNA levels with overweight and with serum levels of leptin and insulin in males, but not in females, and no correlation with serum free fatty acid levels. A subsequent correlation analysis and a multiple linear regression analysis showed that overweight was the only parameter actually related to UCP3 mRNA levels in males. Fasting-induced up-regulation of muscle UCP3 mRNA levels was higher in males (5-7 fold) than in females (3-4 fold). Our results point to the existence of sex-associated differences in the control of muscle UCP3 expression in response to overweight and fasting, with an impaired induction in female rats under both conditions. The correlation of abdominal muscle UCP3 mRNA expression with overweight in males could be related to their relative resistance to gain weight after chronic overeating of a cafeteria diet, by the purported role of UCP3 in the regulation of lipid utilization.