The transcription factor GATA-4 plays an important role in myocardial protection. Astragaloside IV (Ast-IV) was reported with the effects on improving cardiac function after ischemia. In this study, we explored how Ast-IV interacts with GATA-4 to protect myocardial cells H9c2 against Hypoxia/Reoxygenation (H/R) stress. H9c2 cells were cultured under the H/R condition. Various cell activity and morphology assays were used to assess the rates of apoptosis and autophagy. In these H/R injured H9c2 cells, increased apoptosis (P < 0.01) and autophagosome number (P < 0.01) were observed, and the addition of Ast-IV ameliorated this tendency. Mechanistically, we used the RT-qPCR and Western blot to evaluate the expressions of various molecules. The results showed that Ast-IV treatment upregulated gene expression of GATA-4 (P < 0.01) and the survival factors (Bcl-2, P < 0.05; p62, P < 0.01), but suppressed apoptosis and autophagy related genes (PARP, Caspase-3, Beclin-1, and LC3-II; All P < 0.01). Furthermore, overexpressing of GATA-4 by its agonist phenylephrine can also protect H/R injured H9c2 cells, and the addition of Ast-IV further enhanced this protection of GATA-4. In contrast, silencing GATA-4 expression abolished the H/R protection of Ast-IV, which demonstrated that the myocardial protection of Ast-IV is mediated by GATA-4. Lastly, along with GATA overexpression, enhanced interactions between Bcl-2 and Beclin-1 were detected by Chromatin immunoprecipitation (P < 0.01). Ast-IV rescued the H/R injury induced apoptosis and autophagy in H9c2 cells. Ast-IV treatment can stimulate the overexpression of GATA-4, and further enhanced the myocardial protection effect of GATA-4. Copyright © 2020 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.