Exposure to a high-carbohydrate (HC) milk formula during the suckling period results in permanent metabolic programming of hyperinsulinemia in HC rats. Previous studies have shown that hyperinsulinemia in HC rats involves a programmed hyperresponsiveness to glucose. However, the immediate onset and persistence of enhanced insulin secretion throughout life suggests a role for numerous factors that control insulin secretion. Present in vivo and in vitro studies have shown a role for altered autonomic activity, including increased parasympathetic and decreased sympathetic activities, in the maintenance of hyperinsulinemia in 100-day-old HC rats. HC rats were shown to be more sensitive to cholinergic-induced potentiation of glucose-stimulated insulin secretion (GSIS) in response to acetylcholine and showed increased sensitivity to blockade of cholinergic-induced insulin secretion by the muscarinic-type 3 receptor specific antagonist 4-diphenylacetoxy-N-methylpiperidine. In addition, HC rats were less sensitive to adrenergic-induced inhibition of insulin secretion by oxymetazoline, while treatment with yohimbine resulted in increased GSIS. Furthermore, HC rats showed greater reductions in plasma insulin levels after vagotomy, as well as an attenuation of yohimbine-induced potentiation of GSIS, suggesting that yohimbine-mediated changes are mediated by parasympathetic activity. Changes in autonomic regulation of GSIS are supported by increased mRNA levels of the parasympathetic signaling molecules muscarinic-type 3 receptor, phospholipase C-1, and protein kinase C and decreased levels of _2a-adrenergic receptors in islets from adult HC rats. In conclusion, metabolic programming of hyperinsulinemia throughout adulthood of HC rats involves changes in autonomic activity in response to the HC dietary intervention in the suckling period.