Previous studies have demonstrated the protective signaling of hypoxia-inducible factor (HIF)-1 ␣ against ischemia-reperfusion (I/R) injury in the heart. In the present study, we provide further evidence for a cardioprotective mechanism by HIF-1␣ against I/R injury exerted via the mitochondrial protein frataxin, which regulates mitochondrial Fe-S cluster formation. Disruption of frataxin has been found to induce mitochondrial iron overload and subsequent ROS production. We observed that frataxin expression was elevated in mice hearts subjected to I/R injury, and this response was blunted in cardiomyocyte-specific HIF-1␣ knockout (KO) mice. Furthermore, these HIF-1␣ KO mice sustained extensive cardiac damage from I/R injury compared with control mice. Similarly, reduction of HIF-1␣ by RNA inhibition resulted in an attenuation of frataxin expression in response to hypoxia in H9C2 cardiomyocytes. Therefore, we postulated that HIF-1␣ transcriptionally regulates frataxin expression in response to hypoxia and offers a cardioprotective mechanism against ischemic injury. Our promoter activity and chromatin immunoprecipitation assays confirmed the presence of a functional hypoxia response element in the frataxin promoter. Our data also suggest that increased frataxin mitigated mitochondrial iron overload and subsequent ROS production, thus preserving mitochondrial membrane integrity and viability of cardiomyocytes. We postulate that frataxin may exert its beneficial effects by acting as an iron storage protein under hypoxia and subsequently facilitates the maintenance of mitochondrial membrane potential and promotes cell survival. The findings from our study revealed that HIF-1␣-frataxin signaling promotes a protective mechanism against hypoxic/ischemic stress.hypoxia-inducible factor-1␣; frataxin; iron-sulfur; mitochondria; ischemia-reperfusion NEWS & NOTEWORTHYThe present study provides evidence for a cardioprotective transcriptional regulatory mechanism by hypoxia-inducible factor-1␣ of the mitochondrial protein frataxin against ischemia-reperfusion injury. Frataxin regulates mitochondrial Fe-S cluster formation and protects against mitochondrial iron overload, the subsequent ROS production, and myocardial energy dysregulation.