Kidney disease as the result of diabetes places a major economic burden on the Australian population. Current clinical therapies only slow the progression, and therefore, novel therapeutic targets are urgently required. Recent studies from our laboratory have shown a decline in mitochondrial function and impaired oxidative phosphorylation (OxPhos) in the kidney in diabetic nephropathy, leading to excess reactive oxygen species (ROS) production, renal fibrosis and cell death. Here, we used a mouse model of experimental diabetic nephropathy to characterise the function of the mitochondrial permeability transition (mPT) pore, an assembly of proteins that potentiates ROS production and cell death. Ex vivo functional studies of mitochondria isolated from the renal cortex of mice 24 weeks after the induction of diabetes by streptozotocin revealed enhanced susceptibility to mPT pore opening. This led to dissipation of the mitochondrial transmembrane potential and an influx of solutes, causing expansion of the matrix and swelling, as indicated by transmission electron microscopy of renal cortical proximal tubule cells. Loss of the inner mitochondrial membrane potential and the inability to maintain a pH gradient due to proton influx disrupted mitochondrial ATP synthesis, leading to energy depletion and induction of cell death. Intrinsic mitochondrial respiratory capacity by high-resolution respirometry showed basal and ADP-stimulated oxygen consumption to be reduced. This was observed in parallel with increased production of mitochondrial ROS and upregulation of proteins involved in mitochondrial fission and fusion, indicating changes in mitochondrial dynamics. Finally, proof of principle studies in human renal proximal tubule cells demonstrated induction of mPT pore opening by high glucose, which was inhibited by Debio-025, an inhibitor of cyclophilin D, the putative functional pore subunit. This study indicates that the mPT pore is a likely candidate factor conferring susceptibility to diabetic nephropathy and warrants targeting as a new approach to afford superior renoprotection.