The inhibitory action of hyperglycemia is mediated by vagal afferent fibers innervating the stomach and duodenum. Our in vitro studies showed that a subset of nodose ganglia neurons is excited by rising ambient glucose, involving inactivation of ATP-sensitive K⁺ (K_ATP) channels and leading to membrane depolarization and neuronal firing. To investigate whether nodose ganglia K_ATP channels mediate gastric relaxation induced by hyperglycemia, we performed in vivo gastric motility studies to examine the effects of K_ATP channel activators and inactivators. Intravenous infusion of 20% dextrose induced gastric relaxation in a dose-dependent manner. This inhibitory effect of hyperglycemia was blocked by diazoxide, a K_ATP channel activator. Conversely, tolbutamide, a K_ATP channel inactivator, induced dose-dependent gastric relaxation, an effect similar to hyperglycemia. Vagotomy, perivagal capsaicin treatment, and hexamethonium each prevented the inhibitory action of tolbutamide. Similarly, N^G-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, also blocked tolbutamide's inhibitory effect. To show that K_ATP channel inactivation at the level of the nodose ganglia induces gastric relaxation, we performed electroporation of the nodose ganglia with small interfering RNA of Kir6.2 (a subunit of K_ATP) and plasmid pEGFP-N1 carrying the green fluorescent protein gene. The gastric responses to hyperglycemia and tolbutamide were not observed in rats with Kir6.2 small interfering RNA-treated nodose ganglia. However, these rats responded to secretin, which acts via the vagal afferent pathway, independently of K_ATP channels. These studies provide in vivo evidence that hyperglycemia induces gastric relaxation via the vagal afferent pathway. This action is mediated through inactivation of nodose ganglia K_ATP channels.