Insulin secretory dysfunction of the pancreatic β-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A^−/− mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. Thein vivo insulin secretory response to arginine was similar in Rab3A^−/− mice as Rab3A^+/+ control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A^−/− mice, compared with that in Rab3A^+/+ animals, that was markedly increased above that to the first arginine stimulus. There was no difference in β-cell mass or insulin production between Rab3A^−/− and Rab3A^+/+ mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was ∼60–70% lower in Rab3A^−/− islets compared with Rab3A^+/+ controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca²⁺]_i flux between Rab3A^−/− and Rab3A^+/+ islet β-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of β-granules. Also, this study indicates, for the first time, that the in vivoinsulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.