Altitude training is a frequently used method for enhancing endurance performance in athletes. But its acute effect on carbohydrate metabolism in cardiac muscle is unknown. In this study, we determined the acute effect of an exercise-hypoxia challenge on glycogen storage and GLUT4 protein expression in heart muscle. Sixteen male Sprague-Dawley rats were assigned to one of two groups: control (CTRL) and exercise-hypoxia (EX+HY). The exercise protocol consisted of swimming for 180 min twice, with a 45-min rest interval. Five hours after the exercise, the EX+HY rats were exposed to a 14% O(2) systemic hypoxia under normobaric condition for 12 h. After this hypoxia exposure, the EX+HY and control rats were given glucose orally (1 g/kg body weight) with stomach tube and recovered under normal condition for 16 h. Ventricular portion of the heart was used to determine the levels of glycogen, GLUT4 mRNA, and GLUT4 protein after recovery. We found that myocardial glycogen level was lowered by the exercise-hypoxia challenge (51% below control, p < 0.05), while GLUT4 mRNA was dramatically elevated (approximately 400% of the control level, p < 0.05). The acute exercise-hypoxia treatment did not affect GLUT1 protein level in the same tissue. The novel finding of the study was that the exercise-hypoxia treatment significantly induced GLUT4 gene expression in the cardiac muscle. This acute response appears to be associated with a sustained glycogen depletion of the muscle.