Pancreatic -cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of -cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K⁺ (K_V) and Ca²⁺ (Ca_V) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic -cells, and examined their targeting to specialized cholesterol-rich lipid rafts. In -cells, we detected the expression of K_V4.1/4.3 (A- type current), K_V3.2/3.3 (delayed rectifier current), Ca_V1.2 (L-type current), Ca_V2.2 (N-type current), and the SNARE (SNAP-25, syntaxin 1A, VAMP-2) and SNARE-associated proteins (Munc-13-1, Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K_V4.1/4.3, Ca_V1.2, and SNARE proteins (syntaxin 1A, SNAP-25, VAMP-2) target to lipid raft domains on -cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl--cyclodextrin decreased the association of K_V4.1/4.3, Ca_V1.2 and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K_V currents and enhancement of glucagon secretion from -cells. Consistently, capacitance measurements of exocytosis of single -cells showed enhanced exocytosis following membrane cholesterol depletion. Taken together, our results demonstrate the association of K_V4, Ca_V1.2 and SNARE proteins with lipid rafts in pancreatic -cells. Glucagon secretion from -cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.