Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABA receptors is still unknown. Preliminary evidence showed that GABA receptor stimulation can induce an increase in endothelial Ca levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca] that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca release from neutral and acidic Ca stores, respectively, and sustained by store-operated Ca entry. GABA and GABA receptors were both required to trigger the endothelial Ca response. Unexpectedly, we found that the GABA receptors signal in a flux-independent manner via the metabotropic GABA receptors. Likewise, the full Ca response to GABA receptors requires functional GABA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.