Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′- O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2′- O-Me-cAMP-AM was associated with depolarization and an increase of [Ca2+]ithat reflected both Ca2+influx and intracellular Ca2+mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2′- O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis.