RalA is a small GTPase that is thought to facilitate exocytosis through its direct interaction with the mammalian exocyst complex. In this study, we report an essential role for RalA in regulated insulin secretion from pancreatic beta cells. We employed lentiviral-mediated delivery of RalA short hairpin RNAs to deplete endogenous RalA protein in mouse pancreatic islets and INS-1 beta cells. Perifusion of mouse islets depleted of RalA protein exhibited inhibition of both first and second phases of glucose-stimulated insulin secretion. Consistently, INS-1 cells depleted of RalA caused a severe inhibition of depolarization-induced insulin exocytosis determined by membrane capacitance, including a reduction in the size of the ready-releasable pool of insulin granules and a reduction in the subsequent mobilization and exocytosis of the reserve pool of granules. Collectively, these data suggest that RalA is a critical component in biphasic insulin release from pancreatic beta cells.