Glucose-induced insulin secretion from pancreatic β-cells critically depends on the activity of ATP-sensitive K⁺channels (K_ATPchannel). We previously generated mice lackingKir6.2, the pore subunit of the β-cell K_ATPchannel (Kir6.2^−/−), that show almost no insulin secretion in response to glucosein vitro. In this study, we compared insulin secretion by voluntary feeding (self-motivated, oral nutrient ingestion) and by forced feeding (intra-gastric nutrient injection via gavage) in wild-type (Kir6.2^+/+) andKir6.2^−/−mice. Underad libitumfeeding or during voluntary feeding of standard chow, blood glucose levels and plasma insulin levels were similar inKir6.2^+/+andKir6.2^−/−mice. By voluntary feeding of carbohydrate alone, insulin secretion was induced significantly inKir6.2^−/−mice but was markedly attenuated compared with that inKir6.2^+/+mice. On forced feeding of standard chow or carbohydrate alone, the insulin secretory response was markedly impaired or completely absent inKir6.2^−/−mice. Pretreatment with a muscarine receptor antagonist, atropine methyl nitrate, which does not cross the blood–brain barrier, almost completely blocked insulin secretion induced by voluntary feeding of standard chow or carbohydrate inKir6.2^−/−mice. Substantial glucose-induced insulin secretion was induced in the pancreas perfusion study ofKir6.2^−/−mice only in the presence of carbamylcholine. These results suggest that a K_ATPchannel-independent mechanism mediated by the vagal nerve plays a critical role in insulin secretion in response to nutrientsin vivo.