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Protective effect of phosphodiesterase 5 (PDE5) inhibitor sildenafil on hypoxic injury of isolated myocardial cells and its mechanism of action were investigated. Myocardial cells of neonatal mice were isolated, cultured and divided into blank, control, and PDE5 inhibitor group. Cells in the control and the PDE5 inhibitor group were treated with hypoxia and serum deprivation for 6 h to simulate the myocardial ischemia process in vivo, while those in the PDE5 inhibitor group were treated with 1 µmol/l sildenafil. The cell viability was detected via Cell Counting kit-8 (CCK-8), the cytotoxicity was detected via lactate dehydrogenase release assay, and the apoptosis level was detected via flow cytometry, Hoechst staining and caspase-3 activity assay. Moreover, changes in mitochondrial membrane potential of myocardial cells were detected via JC-1 staining and flow cytometry, fluorescein adenosine triphosphate (ATP) assay kit was used to detect the production of ATP in myocardial cells, and reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the level of Sirt3 messenger ribonucleic acid (mRNA) in myocardial cells. Finally, the expression and changes of Sirt3, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and acetylated PGC-1α were detected via western blot analysis. After hypoxia treatment, the cell viability was decreased, the cytotoxic effect was enhanced, the percentage of apoptosis was increased, the activity of apoptosis-related protein was increased, the mitochondrial membrane potential was decreased, the production of ATP was reduced, the expression of Sirt3 was decreased, and the acetylation level of PGC-1α was increased. However, after pretreatment with sildenafil, the damage to membrane potential of myocardial cells was significantly alleviated, and the production of ATP was increased. At the same time, myocardial cell apoptosis was decreased, Sirt3 expression was increased and PGC-1α acetylation was decreased. PDE5 inhibitor inhibits apoptosis of hypoxic myocardial cells through protecting mitochondrial function.