Objective To study the regulatory effect of urocortin (UCN) on ischemia/reperfusion (I/R)-induced myocardial autophagy, so as to explore the myocardial protection mechanism of UCN. Methods Cardiac I/R model was established with rats and hypoxia/reoxygenation(H/R) model was also established with neonatal rat cardiomyocytes. The injury was created by ischemic/hypoxia for 1 h plus reperfusion/reoxygenation for 2 h, and UCN pretreatment was given 1 h before ischemia/hypoxia. The I/R or H/R-induced myocardial injury, myocardial autophagy and autophagy-related gene expression were observed 2 h after reperfusion/reoxygenation. Results UCN pretreatment greatly reduced I/R-induced myocardial damage by decreasing the infarct size, serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentration, increasing the vitality of H/R cardiomyocytes in vitro, and reducing LDH level in the culture supernatant. Moreover, UCN pretreatment also inhibited H/R-induced myocardial autophagy by reducing the ratio of LC3BⅡ/LC3BⅠ and inhibiting expression of autophagy-related genes (Beclin1 and Bnip3). Conclusion UCN can inhibit I/R-induced myocardial autophagy, which may play an important role in the protection against I/R injury.