Mutations of the pancreatic duodenal homeobox gene-1, Pdx1, cause heritable diabetes in humans and mice. A central abnormality with Pdx1 deficiency is increased death of β-cells, leading to decreased β-cell mass. We show that lentiviral suppression of Pdx1 increases death of mouse insulinoma MIN6 β-cells associated with dissipation of the mitochondrial inner membrane electrochemical gradient, Δψ_m. Preventing mitochondrial permeability transition pore opening with the cyclophilin D inhibitor cyclosporin A restored Δψ_m and rescued cell viability. Reduced β-cell mass, markers of β-cell apoptosis, necrosis, and decreased proliferation are present in Pdx1 haploinsufficient mice. Genetic ablation of the Ppif gene, encoding cyclophilin D, restored β-cell mass and decreased TUNEL and complement complex labeling without affecting β-cell proliferation. In adult mice maintained on a high-fat diet, Ppif ablation normalized fasting glucose and glucose and insulin responses to acute glucose challenge. Thus, cyclophilin D and the mitochondrial permeability transition are critical regulators of β-cell death caused by Pdx1 insufficiency.