Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca2+ homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca2+ regulation is bidirectional, depending on the excitability of cells. Under physiological K+/Ca2+ levels, CBD caused a subtle rise in [Ca2+]i, whereas CBD reduced [Ca2+]i and prevented Ca2+ oscillations under high-excitability conditions (high K+ or exposure to the K + channel antagonist 4AP). Regulation of [Ca2+] i was not primarily mediated by interactions with ryanodine or IP3 receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD's [Ca2+]i regulation. Application of carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na +/Ca2+ exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca2+ responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 μm) was neuroprotective against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial dysfunction and Ca2+ dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca2+ homeostasis.