Uncoupling protein 3 (UPC3) is a candidate protein transporter that uncouples oxidative phosphorylation of mitochondrial respiration in skeletal muscle. A number of studies on UCP3 functions under various physiological conditions have suggested that the function of UCP3 is not limited only to regulation of whole-body energy metabolism but is also involved in regulation of substrate (lipids and glucose) metabolism. The purpose of the present study was to clarify the time course of UCP3 mRNA expression in rat skeletal muscle during a 1 h bout of treadmill exercise and to examine whether changes in fat/glucose metabolism modulates UCP3 mRNA expression. The pattern of UCP3 mRNA expression during the exercise was biphasic in both the soleus and gastrocnemius muscles. UCP3 expression increased at 5 min of exercise (soleus: 232%, p < 0.05, gastrocnemius: 185%, p < 0.05, respectively), and at the end of the exercise (196%, p < 0.05 and 193%, p < 0.05, respectively). UCP3 mRNA expression was still increased at 3 h post-exercise in both muscles, 200% (p < 0.05) and 237% (p < 0.05), respectively. However, at 20 min of the exercise, UCP3 mRNA expression was similar to control levels in both muscles (104% and 97%, respectively). The time course of plasma free fatty acid (FFA) did not follow the same time course as UCP3 mRNA expression. Plasma FFA peaked at the end of the exercise, suggesting that FFA did not play a role in inducing UCP3 mRNA expression. Glucose transporter 4 (GLUT4) mRNA expression did not change during or after exercise. These data indicated a rapid acceleration in UCP3’s transcription activity in response to exercise, and suggest that potential factor(s) other than changes in fat/glucose metabolism regulate UCP3 gene expression during moderate exercise.