Objective To investigate the effect of glucose supplement on AMPK activation in myocardium of exercised rats by measuring the myocardial AMPK activation and glycogen content after acute exercise training. Methods Rats were subjected to an acute endurance exercise and glucose supplement in varying doses and time points before and after exercise. The dynamic changes of myocardial AMPK activities was measured with Western blotting, changes of myocardial glycogen content were measured with Anthrone method. Results AMPK activation in myocardium of exercised rat was increased significantly throughout the exercise, and remained at a higher level 1 hour after acute exercise. However the level of AMPK activity was not significantly increased in exercised rat with glucose supplement. Glycogen content was not significantly changed after exercise. Rats subjected to lower dose glucose supplement did not show significant changes in glycogen content neither. But glycogen content was significantly increased in rats at 24 hours after exercise, subjected to higher dose of glucose supplement. Conclusions 1) Acute exercise induces a significant increase in AMPK activation in myocardium of exercised rats. Glucose supplement significantly inhibites the activation of AMPK induced by acute exercise. (2) Higher dose glucose supplement significantly increases glycogen content in the rat myocardium 24 h after exercise.