Myocardial ischemia-reperfusion injury (MIRI) is a major factor that leads to cardiac dysfunction in cardiovascular surgery during extracorporeal circulation. Recent studies have found that ozone (O ₃ ) has protective effect on MIRI caused by the anterior descending branch of the ligated left coronary artery. However, whether O ₃ preconditioning has the same protective effect on global MIRI and the mechanism underlying this clinical treatment remains elusive. Here, we hypothesized that O ₃ preconditioning (O ₃ P) could protect rabbit heart against global MIRI in vitro by up-regulating HIF-1α. Rabbits were treated intraperitoneally with O ₂ /O ₃ mixture with different concentrations and then injected with YC-1 (inhibitor of HIF-1α) before the establishment of the global MIRI model using the Langendorff isolated heart perfusion apparatus. We investigated the effects of O ₃ preconditioning on cardiac systolic function, myocardial infarction, inflammatory response, mitochondrial function, myocardial pathological changes and arrhythmias. We found that the heart with O ₃ preconditioning significantly increased HR, LVDP and IL-10 expression, and decreased IL-6 expression, CK-MB, cTnT and cTnI concentration, myocardial infarction area, myocardial pathological injury and the occurrence of ventricular tachycardia and ventricular fibrillation. Meanwhile, the level of HIF-1α was significantly increased. However, after treatment of specific inhibitor of HIF-1α, the protective effect of O ₃ preconditioning was reversed completely. Our data indicates that O ₃ preconditioning has protective effect on MIRI and this protective effect is positively associated with dosage of O ₃ . Energy metabolism disorder is the initial stage of MIRI and up-regulation of HIF-1α plays an important role in reducing mitochondrial dysfunction.