Objective The purpose of this study was to investigate the expression of mitochondrial motility-related gene mirol and mitochondrial energy metabolism during an acute bout of prolonged exercise. Methods CS7 BL/6 mice underwent a moderate intensity treadmill running with the slope of 0° and at the speed of 13m/min, and they were randomly divided into 5 groups:resting group(R),exercise groups running for 30min(E30),60min(E60),90min (E90) and 120min(E120),respectively. Respiratory control index and ATP synthesis activity in isolated mitochondria were detected. Skeletal muscle H_2O_2 concentration,mirol mRNA expressions were also measured. Results (1)mirol mRNA contents were significantly increased in groups E30 to E120,as compared with the group R;and mirol mRNA expression increased 32.8%, 107.6%,63.8% and 44.8% respectively in groups E30 to E120. (2)H_2O_2 contents of skeletal muscle were clearly increased in group s E30 to E120. (3)ATP synthesis activity elevated at 30 min of exercise and returned to the baseline thereafter,whereas respiratory control index without remarkable change in groups E30 to E120. Conclusion Muscular mirol mRNA expression significantly increased during 120min of exercise as compared with the resting status and thus facilitated the mitochondrial respiratory and ATP synthesis for matching the energy demand during exercise.