The protective effects of sevoflurane post-conditioning against myocardial ischemia/reperfusion (I/R) injury (MIRI) have been previously reported. However, the mechanisms responsible for these protective effects remain elusive. In this study, in order to investigate the molecular mechanisms responsible for the protective effects of sevoflurane post-conditioning on isolated rat hearts subjected to MIRI, Sprague-Dawley rat hearts were randomly divided into the following 6 groups: i) the sham-operated control; ii) 2.5% sevoflurane; iii) ischemia/reperfusion (I/R); iv) 2.5% sevoflurane post-conditioning plus I/R; v) 2.5% sevoflurane post-conditioning + NG-nitro-L-arginine methyl ester (L-NAME) plus I/R; and vi) L-NAME plus I/R. The infarct size was measured using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Additionally, the myocardial nitric oxide (NO), NO synthase (NOS) and nicotinamide adenine dinucleotide (NAD+) levels were determined. Autophagosomes and apoptosomes in the myocardium were detected by transmission electron microscopy. The levels of Bcl-2, cleaved caspase-3, Beclin-1, microtubule-associated protein light chain 3 (LC3)‑I/II, Na+/H+ exchanger 1 (NHE1) and phosphorylated NHE1 protein were measured by western blot analysis. NHE1 mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction. Compared with the I/R group, 15 min of exposure to 2.5% sevoflurane during early reperfusion significantly decreased the myocardial infarct size, the autophagic vacuole numbers, the NHE1 mRNA and protein expression of cleaved caspase-3, Beclin-1 and LC3-I/II. Post-conditioning with 2.5% sevoflurane also increased the NO and NOS levels and Bcl-2 protein expression (p<0.05 or p<0.01). Notably, the cardioprotective effects of sevoflurane were partly abolished by the NOS inhibitor, L-NAME. The findings of the present study suggest that sevoflurane post-conditioning protects the myocardium against I/R injury and reduces the myocardial infarct size. The underlying protective mechanisms are associated with the inhibition of mitochondrial permeability transition pore opening, and with the attenuation of cardiomyoctye apoptosis and excessive autophagy. These effects are mediated through an increase in NOS and a decrease in phopshorylated NHE1 levels.