Astragaloside IV exerts beneficial effects on hypoxia/reoxygenation-induced cardiomyocyte injury. However, the exact mechanisms needs further disclosed. This study was designed to investigate the role of PKCβ/Egr-1 pathway in the protective effect of Astragaloside IV on hypoxia/reoxygenation injury. Exposed under hypoxia/reoxygenation condition, the primary cultured neonatal rat cardiac myocytes viability was reduced significantly. While the expression of Egr-1, the phosphorylated level of PKCβ and ERK kinase, and the activities of SOD and MDA were up-regulated obviously. On the other hand, the cells with Astragaloside IV preconditioning increased cell survival rate compared with hypoxia/ reoxygenation alone. Moreover, the expression of Egr-1, the phosphorylated level of PKCβ, and ERK was decreased in Astragaloside IV group. Taken together, these results suggest that the cardioprotection of Astragaloside IV may be through the down-regulation of PKCβ/Egr-1 pathway.