Myocardial ischemia reperfusion (MI/R) injury is reported to induce apoptosis and autophagy of myocardial cells and contribute to adverse cardiovascular outcomes. Vitexin, a flavonoids compound, exhibited anti-inflammatory, anti-oxidative and antitumor effects in various studies. Here, we investigated the cardioprotective effect and underlying mechanism of vitexin treatment in MI/R injury in vivo and in vitro. We found that vitexin decreased the high cell apoptosis rate caused by MI/R injury significantly in a dose-dependent manner. The expression of cleaved Caspase-3 and Bax was down-regulated and the expression of Bcl-2 was up-regulated by vitexin compared with MI/R model group, indicating that vitexin suppressed apoptosis of rat cardiomyocytes H9c2 cells in MI/R injury. The concentrations of LDH, CK and MDA were decreased while the concentration of SOD was increased by vitexin during MI/R injury. Moreover, vitexin also suppressed autophagy of H9c2 cells in MI/R injury by down-regulating the expression of Beclin1 and LC3I/II while up-regulating the expression of p62. Activation of autophagy by Rapa significantly increased cleaved Caspase-3, Bcl-2 expression and the concentration of LDH, CK, MDA while decreased the concentration of SOD, indicating that vitexin inhibited autophagy to suppress apoptosis of H9c2 cells in MI/R injury. The co-treatment of PI3K inhibitor LY294002 reversed the inhibition on apoptosis and autophagy by vitexin, suggesting that vitexin inhibited apoptosis and autophagy to ameliorate MI/R injury through activation of PI3K/Akt/mTOR signaling pathway. We also constructed MI/R animal model using C57BL/6 mice to investigate the cardioprotective effects of vitexin in vivo. We observed that vitexin mitigated MI/R injury by decreasing the concentrations of LDH, CK and MDA, increasing the concentration of SOD, and counteracting the irregular pathological changes induced by MI/R injury dose-dependently. Vitexin also suppressed apoptosis and autophagy in myocardium cells of C57BL/6 mice in MI/R injury via activation of PI3K/Akt/mTOR signaling pathway. In conclusion, vitexin mitigated MI/R injury by inhibiting apoptosis and excessive autophagy via the PI3K/Akt/mTOR signaling pathway.