Abstract: Cells have two modes of mitochondrial permeability transition (MPT) which produce virtually opposite pathophysiological outcomes of survival or death when responding to apoptotic insults. The transient-MPT (t-MPT) protects mitochondria, whereas the prolonged-MPT (p-MPT), once activated, triggers the ‘point of no return’ for apoptosis or necrosis. Our previous studies show that in addition to scavenging mitochondrial reactive oxygen species, melatonin targets mitochondrial Ca2+ (mCa2+)-mediated MPT for protection during mCa2+-mediated apoptosis in astrocytes. The precise mechanism for how melatonin modulates the MPT during mCa2+ stress, however, remains unelucidated. With the application of fluorescence laser scanning imaging microscopy, this study demonstrated for the first time that melatonin does not inhibit the MPT pore, rather it crucially preserves the pore in its protective mode of t-MPT during mCa2+ stress. Melatonin-preserved t-MPT importantly maintained mitochondrial membrane potential (ΔΨm) which not only prevented depolarized ΔΨm-induced p-MPT but also retained ΔΨm-dependent ATP formation during disturbed Ca2+ homeostasis. Additionally, the melatonin-preserved t-MPT allowed mitochondria to release the toxic overload of mCa2+ to sublethal levels, which prevented mCa2+-mediated fission and mCa2+-dependent p-MPT and possibly also improved mCa2+-dependent ATP synthesis. Melatonin’s effect in reducing the Ca2+ load greatly diminished when the MPT was inhibited by cyclosporine A, suggesting its pore dependency as well as that a preserved t-MPT may be superior to a MPT inhibition in protecting mCa2+-mediated apoptosis. The unique modulation on the MPT provided by melatonin may have extraordinary therapeutic potential in the treatment of mCa2+-mediated astrocyte-associated neurodegenerative pathologies and diseases.