We aimed to study the effect of fentanyl (Fen) preconditioning on cardiomyocyte apoptosis induced by ischemia-reperfusion (I/R) in rats. A total of 120 Sprague Dawley male rats (age: 3 months) were randomly divided into: sham operation group (S group), I/R group, normal saline I/R group (NS group), and fentanyl low, middle, and high dose groups (Fen1: 2 μg/kg; Fen2: 4 μg/kg; Fen3: 6 μg/kg). Heart rate (HR), mean arterial pressure (MAP), left ventricular developed pressure (LVDP), ±dp/dtmax, malondialdehyde (MDA), superoxide dismutase (SOD) activity, creatine phosphokinase-MB (CK-MB), and cardiac troponin-I (cTnI) were measured. Myocardial ischemic (MI) area, total apoptotic myocardial cells, and protein and mRNA expressions of B-cell lymphoma 2 (Bcl-2) and Bax were detected. HR and MAP were higher, while LVDP and ±dp/dtmax were close to the base value in the Fen groups compared to those in the I/R group. Decreased MDA concentration and CK-MB value and increased SOD activity were found in the Fen groups compared to the I/R group, while cTnI concentration was significantly lower in the Fen1 and Fen2 groups (all P<0.05). Myocardial damage was less in the Fen groups compared to the I/R group and the MI areas and apoptotic indexes were significantly lower in the Fen1 and Fen2 groups (all P<0.05). Furthermore, significantly increased protein and mRNA expressions of Bcl-2, and decreased protein and mRNA expressions of Bax were found in the Fen groups compared to the I/R group (all P<0.05). Fentanyl preconditioning may suppress cardiomyocyte apoptosis induced by I/R in rats by regulating Bcl-2 and Bax.