Presynaptic inhibition mediated by G protein–coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gβγ-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca²⁺ channels. Using presynaptic microinjections of botulinum toxins (BoNTs) during paired recordings, we find that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but does not prevent Gβγ-mediated inhibition. In contrast, cleavage of the C-terminal nine amino acids of the 25 kDa synaptosome-associated protein (SNAP-25) by BoNT A prevents Gβγ-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT A blocks the Gβγ inhibitory effect. Finally, removal of the last nine amino acids of the C-terminus of SNAP-25 weakens Gβγ interactions with soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, may represent a target of Gβγ for presynaptic inhibition.