The pancreatic β-cell responds to changes in the nutrient environment to maintain glucose homeostasis by adapting its function and mass. Nutrients can act directly on the β-cell and also indirectly through the brain via autonomic nerves innervating islets. Despite the importance of the brain-islet axis in insulin secretion, relatively little is known regarding its involvement in β-cell proliferation. We previously demonstrated that prolonged infusions of nutrients in rats provoke a dramatic increase in β-cell proliferation in part because of the direct action of nutrients. Here, we addressed the contribution of the autonomic nervous system. In isolated islets, muscarinic stimulation increased, whereas adrenergic stimulation decreased, glucose-induced β-cell proliferation. Blocking α-adrenergic receptors reversed the effect of epinephrine on glucose + nonesterified fatty acids (NEFA)-induced β-cell proliferation, whereas activation of β-adrenergic receptors was without effect. Infusion of glucose + NEFA toward the brain stimulated β-cell proliferation, and this effect was abrogated following celiac vagotomy. The increase in β-cell proliferation following peripheral infusions of glucose + NEFA was not inhibited by vagotomy or atropine treatment but was blocked by coinfusion of epinephrine. We conclude that β-cell proliferation is stimulated by parasympathetic and inhibited by sympathetic signals. Whereas glucose + NEFA in the brain stimulates β-cell proliferation through the vagus nerve, β-cell proliferation in response to systemic nutrient excess does not involve parasympathetic signals but may be associated with decreased sympathetic tone.