G protein–coupled receptors (GPCRs) that couple to G_i/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca²⁺ channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca²⁺ entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABA_B receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT_1b receptors and adrenergic α_2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by G_i/o-coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.