Cannabidiol (CBD), the major nonpsychotropic phytocannabinoid, induces apoptosis in both immortalized and primary lymphocytes and monocytes. However, contrasting effects of CBD on the apoptosis between normal and immortalized glial cells have been reported. This study investigated the proapoptotic effect of CBD on primary microglial cells. Treatment of murine primary microglial cultures with CBD resulted in a time- and concentration-dependent induction of apoptosis, as shown by increase in hypodiploid cells and DNA strand breaks, and marked activation of both caspase-8 and −9. Mechanistic studies revealed that antioxidants, including N-acetyl-L-cysteine and glutathione, the G protein-coupled receptor 55 agonist abnormal-CBD and specific antagonists for vanilloid, and CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl-β-cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD-induced microglial apoptosis and caspase activation. Furthermore, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin-1, all of which were attenuated by MCD. Taken together, these results suggest that CBD induces a marked proapoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin-1. © 2012 Wiley Periodicals, Inc.