Tetrandrine (TET), a bisbenzylisoquinoline alkaloid has been used for the treatment of cardiovascular diseases and hypertension. This study was to investigate whether tetrandrine exerts cardioprotection in ischemia–reperfusion (I/R) injury and the mechanisms involved. The cardioprotection effect and mechanisms of tetrandrine was evaluated by I/R injury cardiac cell model. Hexokinase II (HKII) is the critical regulators of mitochondrial dysfunction in cardiac I/R injury and it participate in the regulation of glycolysis and energy metabolism. The effect of tetrandrine on HKII and Janus kinase (JAK), (Protein kinase B)Akt as well as hypoxia inducible factor α (HIF-α) which are HKII's regulator was also investigated. We found that tetrandrine significantly reduced lactate dehydrogenase, caspase 3 level and apoptosis in I/R injury cardiac cell, meanwhile restored mitochondrial energy metabolism and enhanced glycolysis in model cell. Tetrandrine up-regulated the expression of p-STAT3 and HKII, but has no effect on p-akt and HIF-α. The cardioprotection effect significantly attenuated after tetrandrine combined with JAK3 inhibitor. The expression of p-STAT3 and HK II were also significantly decreased simultaneously. On the contrary, combined with JAK1/2 inhibitor, there was no significant influence. In addition, tetrandrine increased the JAK3 in model cells, but have no impact on the expression of JAK1, JAK2. Taken together, these data revealed that the cardioprotection effect of tetrandrine appears to be involved in the JAK3/STAT3 /HK II.