Orientin, a flavonoid exists in Chinese traditional herbal Polygonum orientale L., has been previously demonstrated to protect against myocardial ischemia reperfusion injury (MIRI) through inhibition of apoptosis. However, the underlying mechanisms remain to be elucidated and we therefore in this study investigated the effects of orientin on autophagy during MIRI in rats. The results indicate that orientin, at the concentrations of 10 and 30 μM in the cultures of neonatal rat cardiomyocytes, promoted the induction of autophagy, increasing the formation of autophagosomes and enhancing the expression of LC3 puncta, LC3-II/LC3-I ratio and Beclin 1 after hypoxia/reoxygenation. The induction of autophagy by orientin correlated with enhanced cell viability and decreased apoptosis, which was significantly attenuated by autophagy inhibitor wortmannin, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Moreover, application of orientin increased the activation of AMPK and Akt, downregulated the phosphorylation of mammalian target of rapamycin (mTOR) and the expression of Raptor, and enhanced the interaction between Beclin 1 and Bcl-2 in endoplasmic reticulum due to increased phosphorylation of Beclin 1 and decreased phosphorylation of Bcl-2. Our investigation suggests that the cardioprotective effects of orientin during MIRI may be mediated through the balance of autophagy through regulating AMPK, Akt, mTOR, and Bcl-2 associated signaling pathways.