Astragaloside IV (ASI), a traditional Chinese medicine, is a main active ingredient of Astragalus membranaceus. Many clinical studies have found that ASI protects cardiomyocytes in cardiovascular diseases, but the underlying mechanisms remain obscure. The aim of this study was to investigate the molecular mechanisms responsible for the protective effects of ASI in cardiomyocytes from anoxia/reoxygenation (A/R) injury. According to the previous studies, we hypothesized that the cardioprotective effects of ASI against A/R injury might be associated with Notch1/Hes1 signaling pathway. In this study, neonatal rat primary cardiomyocytes were preconditioned with ASI prior to A/R injury. Our results showed that ASI effectively increased the cell viability, decreased the content of MDA, decreased the activities of CPK and LDH, increased the activities of GSH-Px and SOD, and reduced the reactive oxygen species (ROS) generation and the loss of mitochondrial membrane potential (Δψm). ASI inhibited the mitochondrial permeability transition pore (mPTP) opening and activation of caspase-3, and finally decreased the cell apoptosis in cardiomyocytes. Furthermore, ASI upregulated Hes1 protein expression. However, pretreatment with DAPT, a Notch1 inhibitor, effectively attenuated the cardioprotective effects of ASI against A/R injury, except MDA, SOD, GSH-Px, and the ROS generation. Taken together, we demonstrated that ASI could protect against A/R injury via the Notch1/Hes1 signaling pathway.