Minocycline has been shown to be neuroprotective in ischemic and neurodegenerative disease models and could potentially be relevant for clinical use. We revisited the hypothesis that minocycline acts through direct inhibition of calcium-induced mitochondrial permeability transition (mPT) resulting in reduced release of cytochrome c (cyt c). Minocycline, at high dosage, was found to prevent calcium-induced mitochondrial swelling under energized conditions similarly to the mPT inhibitor cyclosporin A (CsA) in rodent mitochondria derived from the CNS. In contrast to CsA, minocycline dose-dependently reduced mitochondrial calcium retention capacity (CRC) and respiratory control ratios and was ineffective in the de-energized mPT assay. Further, minocycline did not inhibit calcium- or tBid-induced cyt c release. We conclude that the neuroprotective mechanism of minocycline is likely not related to direct inhibition of mPT and propose that the mitochondrial effects of minocycline may contribute to toxicity rather than tissue protection at high dosing in animals and humans.