Increased oxidative stress and intracellular calcium levels and mitochondrial overloading of calcium during ischemic renal injury (IRI) favor mitochondrial membrane permeability transition pore (MPTP) opening and subsequent necrotic cell death. Cyclophilin D (CypD) is an essential component of MPTP, and recent findings implicate its role in necrotic, but not apoptotic, cell death. To evaluate the role of CypD following IRI, we tested the hypothesis that CypD gene ablation protects mice from IRI. Renal function as assessed by plasma levels of both creatinine and blood urea nitrogen was significantly reduced in CypD knockout (CypD(-/-)) mice compared with wild-type mice during the 5-day post-ischemia period. Erythrocyte trapping, tubular cell necrosis, tubular dilatation, and neutrophil infiltration were significantly decreased in CypD(-/-) mice. To define the mechanisms by which CypD deficiency protect the kidneys, an in vitro model of IRI was employed. Inhibition of CypD using cyclosporin A in oxidant-injured cultured proximal tubular cells (PTC) prevented mitochondrial membrane depolarization, reduced LDH release, ATP depletion and necrotic cell death. Similarly, oxidant-injured CypD(-/-) PTC primary cultures were protected from cytotoxicity and necrosis. To conclude, CypD gene ablation offers both functional and morphological protection in mice following IRI by decreasing necrotic cell death possibly via inhibition of MPTP and ATP depletion.