Introduction: Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. The aim of the present study was to evaluate oxytocin (OT) induced cardioprotection and its signaling pathways on lactate dehydrogenase (LDH) and creatine kinase-MB isoenzyme (CK-MB) in the anesthetized rats. Methods: Eighty-four rats were divided into fourteen groups. Animal's hearts were subjected to 25 min ischemia and 2 h reperfusion (I/R). Oxytocin (0.03 μg/kg) was used 25 min prior to ischemia. Atosiban, an OT receptor antagonist (1.5 μg/kg), 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-dependent potassium channel (10 mg/kg), atractyloside, an opener of mitochondrial permeability transition pores (5 mg/kg), chelytraine, a protein kinase C inhibitor (5 mg/kg) and N-acetylcysteine, a reactive oxygen species scavenger (200 mg/kg) were used 10 min prior to OT administration. Then, LDH and CK-MB levels in plasma were measured. Results: OT administration significantly decreased CK-MB and LDH levels compared to I/R group. Administration of atosiban, 5-hydroxydecanoic, atractyloside, chelytraine and N-acetylcysteine abolished the cardiac preconditioning effect of OT. Conclusion: The present study demonstrates that oxytocin cardioprotective effects are complex and its signaling pathways may mediate through mKATP channels, mPTP, PKC activation and increase ROS