Hydroxysafflor yellow A (HSYA) is the main active component of Carthamus tinctorius L which has been used for hundreds of years in Chinese folk medicine in the treatment cardiovascular disease. This study was designed to investigate whether HSYA exerts cardioprotection in ischemia–reperfusion (I/R) injury heart and the mechanisms involved. The protective effect and mechanisms in myocardial ischemia reperfusion injury of HSYA was evaluated by hypoxia–recover (H/R) injury cell model which induced by hypoxia and recovered with oxygen in H9c2 cells. PI3K/Akt and ERK as the reperfusion injury salvage kinase (RISK) pathway and Hexokinase II (HKII) were both examined. In H/R cell model, HSYA significantly reduced dehydrogenase (LDH), Caspase 3 level, alleviated oxidative stress injury and apoptosis, meanwhile restored mitochondrial energy metabolism. Pretreatment with PI3K inhibitor (LY294002) or hexokinase II inhibitor (3-BrPA), the protective effect of HSYA was significantly attenuated. On the contrary, pretreatment with ERK inhibitor (PD98059), the protective effect of HSYA on myocardial cells was decreased slightly, not as significant as PI3K inhibitor or hexokinase II inhibitor. ERK play a protective role in myocardial protection by phosphorylation of GSK3-β, but the effect of HSYA on phosphorylation of GSK3-β is weakly, however the effect of HSYA on Akt and hexokinase II were significantly up-regulated. Meanwhile, the phosphorylation of GSK3-β by HSYA was significantly reduced after gave the ERK inhibitor and had no significant difference between the model group. The cardioprotection effect of HSYA appears to be mainly mediated via the PI3K/Akt/hexokinase II.