Objective: To investigate the mechanism of astragaloside IV(As-IV) on alleviation of hypoxia/reoxygenation induced myocardial injury. Methods: Cultured cardiomyocytes from neonatal SD rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation(H/R). Meanwhile,As-IV (30 -滋mol/L) was treated in H/R cardiomyocytes. Myocytes injury was determined by the release of creatine kinase (CK-MB) in supernatant. Myocardial SERCA2a activity was measured,PKA C subunit α (PKA-Cα) gene expression and Ser16 phosphorylated phospholamban(Ser16-PLN) protein expression level were detected by real-time PCR and Western blot respectively. Results: Cultured cardiomyocytes exposed to hypoxia/reoxygenation presented statistically higher CK-MB in supernatant,decreased myocardial SERCA2a activity,reduced PKA-Cα gene and Ser16-PLN protein expressions(P < 0.05). But As-IV treatment significantly prevented the alterations in H/R cardiomyocytes mentioned above. Conclusion: These results suggest that the cardioprotective effects of As-IV may be associated with upregulation of PKA-Cα gene and Ser16-PLN protein expressions,thus restoring the SERCA2a function in hypoxia/reoxygenation injury.