The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia-reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma-Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia-reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.