Piceatannol (3,3′,4,5′-trans-trihydroxystilbene) is a natural polyphenols compound that occurs hydroxylated analogue of resveratrol showing widely biological activities. Previous studies have demonstrated its functions on anti-cancer, neuroprotection and cardioprotection. However, few studies have clarified the benefits of piceatannol on cardiomyocytes except its anti-oxidative effect based on the original property of polyphenols. Here we apply H9c2 cardiomyocytes to study the cardioprotective mechanisms of piceatannol in vitro. We firstly verify its anti-peroxidation effect by using H₂O₂-induced in vitro model. Then, flow cytometry results show piceatannol reduce cellular apoptosis by enhancing Bcl-2 expressions in immunoblot analysis. Meantime, piceatannol decreases H₂O₂-induced excessive ROS and calcium overloading, and prevents mitochondrial depolarization. Most importantly, piceatannol pretreatment can regulate PI3K-Akt-eNOS signaling pathway to alleviate peroxidative injury. Immunoblot analysis of PI3K, Akt, p-Akt and eNOS shows H₂O₂ significantly reduces expressions of these proteins. Pretreatment of piceatannol evidently increases their expressions and decreases iNOS expression, implying piceatannol can upregulate PI3K-Akt-eNOS signaling to protect cardiomyocytes from peroxidative injury.