Background Renal ischemia–reperfusion injury (IRI) remains a leading cause of acute kidney injury (AKI) worldwide and contributes significantly to postoperative renal failure and graft dysfunction in transplant recipients. The pathogenesis of IRI is largely mediated by excessive generation of reactive oxygen species (ROS) and mitochondrial injury, which together initiate inflammation, cell death, and loss of renal function. This study investigated the role of oxidative stress and mitochondrial dysfunction in renal IRI and evaluated the protective efficacy of the mitochondria-targeted antioxidant MitoQ in experimental and clinical settings in South Africa. Methods A combined experimental and translational study design was employed. Forty-eight male Wistar rats were divided into four groups: Sham, IRI, IRI + N-acetylcysteine (NAC), and IRI + MitoQ. Renal ischemia was induced by bilateral clamping of renal pedicles for 45 min followed by 24 h of reperfusion. Biochemical markers of oxidative stress (MDA, GSH, SOD, and 8-OHdG), mitochondrial function (ATP content, membrane potential, OCR), and protein expression (Drp1, MFN2, PGC-1α) were assessed. The clinical arm included forty adult patients undergoing partial nephrectomy or kidney transplantation at Groote Schuur Hospital, where pre- and post-reperfusion renal biopsies and plasma samples were analyzed for oxidative and mitochondrial markers. Statistical analysis used one-way ANOVA, Pearson correlations, and p < 0.05 as the significance threshold. Results The IRI group demonstrated marked oxidative stress, with a threefold rise in malondialdehyde and significant depletion of GSH and SOD (p < 0.001). Mitochondrial dysfunction was evidenced by decreased oxygen consumption, loss of membrane potential, reduced ATP production, and upregulation of Drp1 alongside downregulation of MFN2 and PGC-1α. Pretreatment with MitoQ significantly attenuated lipid peroxidation, restored antioxidant enzyme activity, preserved mitochondrial architecture, and normalized gene expression of Nrf2, HO-1, SOD2, and PGC-1α (p < 0.01 vs. IRI). Histopathology confirmed substantial reduction in tubular necrosis and inflammatory infiltration. Human renal biopsies mirrored these findings, showing increased oxidative and mitochondrial injury after reperfusion, which was reduced in patients receiving perioperative antioxidant supplementation. Conclusions Renal IRI results from a vicious cycle of oxidative stress and mitochondrial failure, culminating in energy depletion and tubular necrosis. MitoQ provided superior renoprotection compared to conventional antioxidants, highlighting the therapeutic potential of targeting mitochondrial ROS generation. These findings provide translational evidence that preserving mitochondrial integrity is central to mitigating renal ischemic injury, particularly in high-risk surgical and transplant populations within South Africa.