Extracellular nucleotides modulate several cell functions via specific receptors, P2X and P2Y. We explored the differential role of these receptors in the control of insulin secretion (InSec). In INS-1e cells grown in 11 mM glucose and then acutely exposed to 3.3, 7.5, 11, or 20 mM, coincubation with ATP, the global agonist of both P2X and P2Y receptors, induced a dose-dependent (p<0.0001) reduction in insulin release (p<0.0001) that was more marked at higher glucose concentrations (p<0.0001 for the interaction). This effect was fully prevented (p<0.0001) by incubating ATP-treated cells in the presence of apyrase, an ecto-ATP/ADPase. Uridine 5'-triphosphate (UTP), preferential agonist of P2Y receptors, significantly stimulated InSec at all glucose concentrations tested, while benzoyl-benzolyl ATP (BzATP), strong and highly selective P2X₇ agonist, did not influence InSec. Oxidized-ATP, which completely suppresses P2X activity, abolished the inhibitory effect of ATP on InSec. Similar results were obtained in MIN-6 cells. Stimulation with ATP, BzATP, and UTP dose-dependently increased [Ca²⁺]_i. By riRNA we shown P2X₃ and P2Y₄ as the main responsible of inhibitory and promoting effect on InSec, respectively. As P2X₇ is not directly involved in InSec, we tested whether the effect of ATP on hormone synthesis might be mediated by apoptosis. However, neither ATP nor BzATP induced either early or late apoptosis. We conclude that (a) INS-1e cells express multiple purinergic receptors, (b) ATP reduces glucose-induced InSec as a net effect of inhibition through P2X and stimulation through P2Y receptors, and (c) P2X-mediated apoptosis is not involved in the inhibition of InSec.