Rho GDP-dissociation inhibitor (GDIα) inhibits glucose-stimulated insulin secretion (GSIS) in part by locking Rho GTPases in an inactive GDP-bound form. The onset of GSIS causes phosphorylation of GDIα at Ser174, a critical inhibitory site for GDIα, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIα in islet β cells remains elusive. We propose that SAD-A, a member of AMP-activated protein kinase-related kinases that promotes GSIS as an effector kinase for incretin signaling, interacts with and inhibits GDIα through phosphorylation of Ser174 during the onset GSIS from islet β cells. Coimmunoprecipitation and phosphorylation analyses were carried out to identify the physical interaction and phosphorylation site of GDIα by SAD-A in the context of GSIS from INS-1 β cells and primary islets. We identified GDIα directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIα complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIα phosphorylation at Ser174 in response to GSIS, which is dramatically potentiated by glucagonlike peptide-1, an incretin hormone. Conversely, SAD-A deficiency, which is mediated by short hairpin RNA transfection in INS-1 cells, significantly attenuated endogenous GDIα phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIα on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIα at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.