Glucagon-producing α-cells are the second most abundant cell type in the islet. While α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphologic and functional maturation occurs to form adult islets. To determine if α-cells have a role in postnatal islet development, a diphtheria toxin mediated α-cell ablation mouse model was established. Rapid and persistant depletion of α-cells was achieved by daily injection of the toxin for 2 weeks starting at post-natal day 1 (P1). Total pancreatic glucagon content in the α-cell ablated mice was undetectable at P14, and still less than 0.3% of that of the control mice at 4 months of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near total lack of α-cells. Furthermore, there were no differences in expression of β-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis nor functional maturation of β-cells.