Extant studies have implicated the Rho subfamily of guanosine triphosphate-binding proteins (G-proteins; e.g., Rac1) in physiological insulin secretion from isolated beta-cells. However, very little is known with regard to potential regulation by G-protein regulatory factors (e.g., the guanosine diphosphate-dissociation inhibitor [GDI]) of insulin secretion from the islet beta-cell. To this end, using Triton X-114 phase partition, co-immunoprecipitation, and sucrose density gradient centrifugation approaches, we report coexistence of GDI with Rac1 in insulin-secreting beta-cells (INS cells). Overexpression of wild-type GDI significantly inhibited glucose-induced, but not KCl- or mastoparan-induced, insulin secretion from INS cells. Furthermore, glucose-stimulated insulin secretion (GSIS) was significantly increased in INS cells in which expression of GDI was inhibited via the small interfering RNA-mediated knockdown approach. Together, these data appear to suggest an inhibitory role for GDI in the glucose metabolic signaling cascade, which may be relevant for GSIS.