SNAP-25 is important for Ca²⁺-dependent fusion of Large Dense Core Vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as GLP-1 and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25 and cAMP-dependent stimulation of insulin exocytosis we have used capacitance measurements, protein-binding assays and Western blot analysis. In insulin secreting INS-1 cells overexpressing wild-type SNAP-25 (SNAP-25_WT) rapid exocytosis was stimulated >3-fold by cAMP, similar to the situation in non-transfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-25_1-197) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the Readily Releasable Pool (RRP), especially from the Immediately Releasable Pool (IRP), whereas cAMP enhanced mobilization of granules from the Reserve Pool (RP) in both SNAP-25_1-197 (P<0.01) and SNAP-25WT (P<0.05) cells. This was supported by hormone release measurements. Augmentation of IRP by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus, we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin secreting cells.