Acylated and unacylated ghrelin (AG and UAG, respectively) are gut hormones that exert pleiotropic actions, including regulation of insulin secretion and glucose metabolism. In this study we investigated whether AG and UAG differentially regulate portal and systemic insulin levels after a glucose load. We studied the effects of the administration of AG (30 nmol/kg), UAG (3 and 30 nmol/kg), the ghrelin receptor antagonist [D-Lys³]GHRP-6 (1 µmol/kg), or various combinations of these compounds on portal and systemic levels of glucose and insulin after an intravenous glucose tolerance test (IVGTT, D-glucose 1g/kg) in anesthetized fasted Wistar rats. UAG administration potently and dose-dependently enhanced the rise of insulin concentration induced by IVGTT in the portal and, to a lesser extent, in the systemic circulation. This UAG-induced effect was completely blocked by the co-administration of exogenous AG at equimolar concentrations. Similarly to UAG, the ghrelin receptor antagonist [D-Lys³]GHRP-6, alone or in combination with AG and UAG, strongly enhanced the portal insulin response to IVGTT, whereas exogenous AG alone did not exert any further effect. Our data demonstrate that in glucose-stimulated conditions exogenous UAG acts as a potent insulin-secretagogue, whereas endogenous AG exerts a maximal tonic inhibition on glucose-induced insulin release.