Parishins, important constituents of Gastrodia elata (G. elata), are known to exhibit a number of biological and pharmacological properties. However, their role and mechanisms of action in myocardial ischemia are unknown. The present study investigated the potential protective effects and mechanisms of parishins extracted from G. elata on hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes. The results demonstrated that parishins had significant protective effects on myocardial cells with parishins J and B providing greater cardioprotection through down-regulation of the level of cleaved-caspase-3 and cytochrome c in the cytoplasm and Bax, and up-regulation of cytochrome c in the mitochondria and Bcl-2 than induced by the positive control gastrodin. Additional study of the mechanisms of action indicated that the myocardial protection provided by parishin J was due to inhibition of JNK1 phosphorylation levels, down-regulation of c-jun and ATF-2 phosphorylation levels, a decrease in the phosphorylation of 14-3-3 and an increase in its binding to Bax. Therefore, parishin J was revealed to be a promising candidate as a novel treatment for myocardial protection.