ABSTRACT The permeability transition pore (PT-pore) mediates cell death through the dissipation of the mitochondrial membrane potential (ΔΨm). Because the exact composition of the PT-pore is controversial, it is crucial to investigate the actual molecular constituents and regulators of this complex. We found that mitochondrial creatine kinase-1 (CKMT1) is a universal and functionally necessary gatekeeper of the PT-pore, as its depletion induces mitochondrial depolarization and apoptotic cell death. This can be inhibited efficiently by bongkrekic acid, a compound that is widely used to inhibit the PT-pore. However, when the ‘classical’ PT-pore subunits cyclophilin D and VDAC1 are pharmacologically inhibited or their expression levels reduced, mitochondrial depolarization by CKMT1 depletion remains unaffected. At later stages of drug-induced apoptosis, CKMT1 levels are reduced, suggesting that CKMT1 downregulation acts to reinforce the commitment of cells to apoptosis. A novel high-molecular-mass CKMT1 complex that is distinct from the known CKMT1 octamer disintegrates upon treatment with cytotoxic drugs, concomitant with mitochondrial depolarization. Our study provides evidence that CKMT1 is a key regulator of the PT-pore through a complex that is distinct from the classical PT-pore.