Puerarin (Pur), which is the major bioactive ingredient extracted from the root of Pueraria lobata (Willd.) Ohwi, has been demonstrated to relieve myocardial ischemia/reperfusion (I/R) injury. Macroautophagy, or autophagy, is an evolutionarily conserved cellular catabolic mechanism that is involved in myocardial I/R injury. The present study evaluated the involvement of autophagy in the protective mechanisms of Pur during myocardial hypoxia/reoxygenation (H/R). The results revealed that Pur and 3‑methyladenine pretreatment exerted a cardioprotective effect against H/R‑induced cell viability loss. Pur also decreased the ratio of light chain 3 (LC3) ‑II/LC3‑I and the degradation of p62 during H/R, which was accompanied by an increased level of phosphorylated‑protein kinase B (Akt). These findings suggested that autophagy during myocardial H/R was inhibited by Pur, and this was further confirmed by the results of transmission electron microscopy and adenovirus‑monomeric red fluorescent protein‑green fluorescent protein‑light chain 3 transfection. Furthermore, Pur inhibited the increased levels of autophagy induced by rapamycin, and the autophagy‑inhibiting effects of Pur during myocardial H/R were abolished by the Akt signaling inhibitor API‑2. Collectively, these data indicate that Pur pretreatment may attenuate myocardial H/R injury by inhibiting autophagy via the Akt signaling pathway.