How to provide effective prevention and treatment of myocardial ischemia/reperfusion (I/R) injury and study of the mechanism underlying I/R injury are hotspots of current research. This study aimed to elucidate the effect and cardioprotective mechanism of vitamin C (VC) on myocardial I/R injury. Our study introduced two different I/R models: I/R in vitro and oxygen-glucose deprivation/recovery (OGD/R) in primary neonatal rat cardiac myocytes. We used the mitochondrial permeability transition pore (mPTP) opener lonidamine (LND) and the mitochondrial KATP (mitoKATP) channel inhibitor 5-hydroxydecanoate (5-HD) to analyze the underlying mechanisms. We found that post-treatment with VC decreased I/R injury in our models. Post-treatment with VC significantly decreased I/R-induced injury, attenuated apoptosis, and maintained the functional integrity of mitochondria via alleviation of Ca(2+) overload, reactive oxygen species burst, inhibition of the opening of mPTP, and prevention of mitochondrial membrane potential (ΔΨm) depolarization. VC post-treatment increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3β. The present results demonstrate that VC might protect the myocardium from I/R-induced injury by inhibiting the mPTP opening via activation of mitoKATP channels. VC mediates cardioprotection via activation of the phosphatidyl inositol 3-kinase (PI3K)-Akt signaling pathway. These findings may contribute toward the development of novel strategies for clinical cardioprotection against I/R injury.