This study explores the potential protective effects and mechanisms of astragaloside (AST) on microwave radiation-induced cardiac injury. Rats and H9c2 cells were irradiated with S-band microwave to induce in vivo and in vitro cardiac injury models. In irradiated rats, experiments such as electrophysiological examination, serum biochemical analysis, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), western blot, and immunohistochemical staining were performed after AST were administrated for 7 and/or 14 days. In irradiated H9c2 cells that were pretreated with 1-Azakenpaullone (glycogen synthase kinase-3b inhibitor) or AST, experiments such as TEM, cell counting kit-8 assay, western blot, tetramethylrhodamine methylester staining, and determination of reactive oxygen species (ROS), adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) were performed. In vivo results showed that at 7 days after exposure, microwave radiation-induced severe cardiac injury (as evidenced by abnormal electrocardiograms and cardiac tissue structure, increased serum myocardial enzyme activities and Ca²¹ concentration) and lower level of phosphorylation of glycogen synthase kinase-3b (p-GSK-3b^Ser9). All these changes were reversed after AST treatment. The results of in vitro experiments showed that microwave radiation induced a lower level of p-GSK-3b^Ser9, more mitochondrial permeability transition pore (mPTP) opening and more serious mitochondrial dysfunction (characterized by increased intracellular ROS production, decreased intracellular ATP synthesis and MMP decline) in H9c2 cells. All these changes were reversed by 1-Azakenpaullone and AST pretreatment. The findings suggest that AST could shield against microwave radiation-induced cardiac injury by promoting the phosphorylation of GSK-3b^Ser9, thereby inhibiting mPTP opening and restoring mitochondrial function. This study offers valuable insights into potential therapeutic strategies for mitigating the adverse effects of microwave radiation on cardiac health.