Abstract: Melatonin protects cells against various types of oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological and disease condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS indiscriminately damage mitochondrial components and more importantly they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. This study, thus, investigated this possibility of an ‘oxidation free Ca2+ stress’ in the presence of vitamin E after ionomycin exposure as a sole Ca2+-mediated MPT in order to exclude melatonin’s primary antioxidative effects as well as Ca2+-mediated oxidative stress. The studies were carried out using cultured rat brain astrocytes RBA-1. With the application of laser scanning multiple fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protective effects provided by melatonin during Ca2+ stress. First, melatonin, due to its primary antioxidative actions, completely prevented mCa2+-induced mROS formation during ionomycin exposure. Secondly, when melatonin’s antioxidative effects were prevented due to the addition of vitamin E, melatonin significantly prevented mCa2+-mediated MPT and apoptosis suggesting its direct targeting of the MPT. Surprisingly, in the presence of cyclosporin A, a MPT inhibitor, melatonin reduced further mCa2+-mediated apoptosis during ionomycin exposure also suggesting its targeting beyond the MPT. As astrocytes are actively involve in regulating synaptic transmission and neurovascular coupling in the CNS, these multiple mitochondrial layers of protection provided by melatonin against mCa2+-and/or mROS-mediated apoptosis in astrocytes may be crucial for future therapeutic prevention and treatment of astrocyte-mediated neurodegenerative diseases in the CNS.