Insulin secretion is initiated by ionic events involving membrane depolarization and Ca2+ entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the β-cell voltage-dependent K+ channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Ky2.1. SNAP-25 reduced currents from recombinant Ky2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K+ currents from rat β-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward KK+ currents in β-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other β-cell Kv channels. This study demonstrates that SNAP-25 can regulate Ky2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.