Resveratrol is a stilbene present in different plant species and exerting numerous beneficial effects, including prevention of diabetes and attenuation of some diabetic complications. Its inhibitory effect on insulin secretion was recently documented, but the exact mechanism underlying this action remains unknown. Experiments employing diazoxide and high concentration of K+ revealed that in depolarized pancreatic islets incubated for 90 min with resveratrol (1, 10 and 100 µM) insulin secretion stimulated by glucose and leucine was impaired. The attenuation of the insulin secretory response to 6.7 mM glucose was not abrogated by blockade of intracellular estrogen receptors and was found to be accompanied by diminished islet glucose oxidation, enhanced lactate production and reduced ATP levels. Glucose-induced hyperpolarization of mitochondrial membrane was also reduced in the presence of resveratrol. Moreover, in depolarized islets incubated with 2.8 mM glucose activation of protein kinase C or protein kinase A potentiated insulin release, however, under these conditions, resveratrol was ineffective. Further studies also revealed that under conditions of blocked voltage-dependent calcium channels the stilbene reduced insulin secretion induced by a combination of glucose with forskolin. These data demonstrate that resveratrol 1) inhibits the amplifying pathway of insulin secretion; 2) exerts insulin-suppressive effect independently on its estrogenic/antiestrogenic activity; 3) shifts islet glucose metabolism from mitochondrial oxidation to anaerobic; 4) fails to abrogate insulin release promoted without metabolic events; 5) does not suppress hormone secretion as a result of the direct inhibition of Ca2+ influx through voltage-dependent calcium channels.