The present study aimed to investigate the cardioprotective effects of acteoside (AC) on myocardial ischemia‑reperfusion injury (MIRI). To meet this aim, a network pharmacological analysis was conducted to search for key genes and signaling pathways associated with AC and MIRI. The infarct size of the rat heart was evaluated using 2,3,5‑triphenyltetrazolium chloride staining, and the serum levels of creatine kinase MB isoenzyme, cardiac troponin I, malondialdehyde and superoxide dismutase were subsequently detected in an in vivo experiment. The inhibitory effect of AC on oxidative stress was further confirmed by assessing the intracellular accumulation of reactive oxygen species (ROS). Hematoxylin and eosin staining was subsequently carried out to observe cardiac histopathological damage. The anti‑apoptotic effects of AC were determined using terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling assay and Hoechst 33342 staining, and the expression levels of apoptosis‑associated proteins in the myocardial tissue were assessed using immunohistochemical analysis. In addition, cell viability was determined using a Cell Counting Kit‑8 assay, and the expression levels of key target proteins associated with AC and MIRI were detected by western blot analysis. The results suggested that pretreatment with AC could mitigate MIRI‑induced myocardial damage, oxidative stress and apoptosis. The anti‑apoptotic effects of AC were associated with elevated Bcl‑2 levels, and reduced caspase‑3 and Bax expression levels in myocardial tissue. In vitro, AC pretreatment both led to an increased rate of cell survival and alleviated oxidative stress, as demonstrated by a decreased level of intracellular ROS accumulation. Moreover, guided by the network pharmacological analysis, heat‑shock protein 90AA1 (HSP90AA1) and the phosphoinositide 3‑kinase (PI3K)/serine‑threonine protein kinase (Akt) signaling pathway emerged as key targets for the action of AC against MIRI. Furthermore, the western blot analysis results showed that pretreatment with AC led to a significant increase in the activity of the PI3K/Akt signaling pathway, in addition to increased expression levels of glycogen synthase kinase‑3β and HSP90AA1. Taken together, the findings of the present study revealed that AC may exert cardioprotective effects on MIRI through suppressing apoptosis and oxidative stress by regulating the expression and activity of key proteins.