Catalpol, a bioactive component from Rehmannia glutinosa (Di Huang), has been widely used to protect cardiomyocytes against myocardial ischemia. The aim of the present study was to investigate the anti-apoptotic and anti-oxidative effects of Catalpol on glucose-starved H9c2 cells for cardio-protection and to elucidate the underlying mechanisms. Here, we showed that Catalpol protected the glucose-starved H9c2 cells through reducing apoptosis and attenuating oxidative damage. Moreover, the increases of autophagic lysosomes, LC3, autophagic flux and autophagic vacuole were observed in Catalpol-treated cells using flow cytometer and fluorescence microscope. Western blotting analyses showed that the autophagy-related proteins (LC3, Beclin1 and ULK) were markedly increased in Catalpol-treated cells, suggesting that Catalpol up-regulated autophagy in glucose starved H9c2 cells. Mechanistic investigations revealed that the autophagy inhibitor 3-MA markedly abrogated Catalpol’s anti-apoptotic and anti-oxidative effects and prevented Catalpol-induced mitophagy. Furthermore, the estrogen receptor inhibitor tamoxifen significantly abolished Catalpol up-regulation of mitophagic related proteins (LC3, Beclin 1, p62, ATG5). Collectively, these data revealed that Catalpol inhibited apoptosis and oxidative stress in glucose-deprived H9c2 cell through promoting cell mitophagy and modulating estrogen receptor, supporting the notion that Catalpol could be a novel drug candidate against myocardial ischemia for the treatment of cardiovascular diseases.