Background−In various cardiovascular diseases, activation of reactive oxygen species-producing enzyme systems lead to increased reactive oxygen species (ROS) and accumulation of their end products, which play important roles in deterioration of the cardiac function. Xanthine oxidase (XO) that is capable of producing a ROS has been designated as a culprit for ischemia/ reperfusion injury of cardiomyocytes. The aims of this study were to evaluate allopurinol a specific inhibitor of XO might play a protective role in ischemia/reperfusion injury. Methods and Results−We investigated the production of ROS, and the concentration of intracellular calcium in various level of XO activation in vitro under the condition of hyoxia/reoxygenation, using rat neonatal cardiomyocytes. We also estimated the effect of allopurinol on the production of ROS, the concentration of intracellular calcium and mitogen-activated protein kinases such as Extracelluar signal-related kinase (ERK) and p38 after treatment with allopurinol of 1, 10µM. Hypoxia/reoxygenation injury of rat cardiomyocytes resulted in activation of XO compared with that of the control (1.10 짹 0.03 vs 0.31 짹 0.05 X 10-4 unit/ µg protein, p<0.05). Allopurinol treatment suppressed hypoxia/reoxygenation injury induced activity of XO (1.10 짹 0.03 vs 0.78 짹 0.05, 0.57 짹 0.02 X 10-4 unit/ µg protein) and the production of ROS (11.33 짹 0.05 vs 10.67 짹 0.05, 10.37 짹 0.03U, p<0.05) with dose-response relationship. Enhanced XO activity augmented concentration of intracelluar Ca2+, allopurinol also had effect on decrease concentration of intracellular Ca2+(30.4 짹 0.05 vs 4.5 짹 0.02 Fluorescence increase %, p<0.05). Enhanced XO activity resulted in activation of phosphorylation of ERK and p38, and these were suppressed by allopurinol treatment. Conclusion−Our studies indicated that under hypoxia/reoxygenation condition, XO activation might be one of the major pathway of producing ROS, and allopurinol would reverse this effect. Therefore it could be demonstrated that allopurinol, xanthine oxidase inhibitor, would play a protective role to cardiomyocytes against ischemia/reperfusion injury.