Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to G??-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of G?? subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that G?? binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, G??, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled G?? interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with G?? for SNARE-binding sites in lipid environments. Mutant G?? subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type G?? were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant G?? subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on G? and G? shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, G?? inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the G??-SNARE interaction and show that the target of G??, downstream of VGCC, is the membrane-embedded SNARE complex.