Myocardial infarction (MI) is the leading cause of death worldwide, and oxidative stress is part of the process that causes MI. Calycosin, a naturally occurring substance with cardioprotective properties, is one of the major active constituents in Radix Astragali. In this study, effect of Calycosin was investigated in vivo and in vitro to determine whether it could alleviate oxidative stress and oxidative stress‐induced cardiac apoptosis in neonatal cardiomyocytes (NCMs) via activation of aldehyde dehydrogenase 2 (ALDH2). Calycosin protected against oxidative stress and oxidative stress‐induced apoptosis in NCMs. Molecular docking revealed that the ALDH2‐Calycosin complex had a binding energy of −9.885 kcal/mol. In addition, molecular docking simulations demonstrated that the ALDH2‐Calycosin complex was stable. Using BLI assays, we confirmed that Calycosin could interact with ALDH2 (KD = 1.9 × 10−4 M). Furthermore, an ALDH2 kinase activity test revealed that Calycosin increased ALDH2 activity, exhibiting an EC50 of 91.79 μM. Pre‐incubation with ALDH2 inhibitor (CVT‐10216 or disulfiram) reduced the cardio‐protective properties Calycosin. In mice with MI, Calycosin therapy substantially reduced myocardial apoptosis, oxidative stress, and activated ALDH2. Collectively, our findings clearly suggest that Calycosin reduces oxidative stress and oxidative stress‐induced apoptosis via the regulation of ALDH2 signaling, which supports potential therapeutic use in MI.