Live-cell imaging experiments were performed with the fluorescent Ca2+ and Zn2+ probes Fluo-4 and FluoZin-3 on cultured cortical neurons dissociated from embryonic mice to investigate the effects of the cannabinoids anandamide (AEA), cannabidiol (CBD), and N-arachidonoyl glycine (NAGly) on neuronal store-operated Ca2+ entry (SOCE). When tested individually AEA, CBD or NAGly inhibited SOCE. CBD and NAGly also released Ca2+ from the endoplasmic reticulum. Furthermore, NAGly mobilized Zn2+ from a store distinct from the endoplasmic reticulum and mitochondria, and up-regulated the thapsigargin-evoked Ca2+ release. All these effects developed in a cannabinoid receptor CB1/2 independent manner via an intracellular pathway sensitive to the GPR55 antagonist ML193. Evidence is presented that cannabinoids influence Ca2+ and Zn2+ signaling in central nervous system neurons. The lipid sensing receptor GPR55 seems to be a central actor governing these responses. In addition, the alteration of the cytosolic Zn2+ levels produced by NAGly provides support for the existence of a connection between endocannabinoids and Zn2+ signaling in the brain.