Assembly of neuronal SNARE protein complexes is essential for fusion of synaptic vesicles with the presynaptic plasma membrane, which releases neurotransmitters into the synaptic cleft and mediates neurotransmission. However, despite the potential of pharmacological regulation of this process for the treatment of various neurological disorders, only a few reagents, including botulinum neurotoxins, are currently available. Here, we report that buforin-1, an antimicrobial peptide from the Asian toad Bufo gargarizans, inhibits neuronal SNARE complex assembly, resulting in neuronal SNARE-mediated membrane fusion in vitro via its direct association with neuronal t-SNAREs syntaxin-1 and SNAP-25. Consistently, buforin-1 significantly inhibited neuronal-SNARE-mediated exocytosis in PC-12 cells. Thus, buforin-1 has potential for the treatment of neurological disorders caused by dysregulated neurotransmission.