Reestablishment of the structural and functional microvasculature would be beneficial to promote healing of diabetic wounds. We explored the role of insulin application on microvascular maturation of diabetic wounds to determine whether it is associated with insulin-induced wound healing. We adopted the multiple injections of streptozotocin (STZ) to establish a diabetic animal model. The effect of insulin on microvessel formation, especially the effect of insulin on microvascular maturation was observed by transmission electron microscopy and laser scanning confocal microscopy. The pivotal protein regulated by insulin during healing processes was explored by tropical application neutralizing antibodies to these proteins; the specific protein was further confirmed using immunoblotting. On days 7 and 11, the blood vessel in insulin-treated wounds was surrounded by more α-smooth muscle actin (α-SMA) expressing cells. The blockage of angiopoietin-1 (Ang-1), but not angiopoietin-2 (Ang-2) or platelet-derived growth factor-B (PDGF-B), resulted in reduced maturation of newly formed blood vessels despite the presence of insulin in vivo. Further analysis showed that insulin induced an increased expression of Ang-1. The blood vessels in insulin-treated wounds showing advanced coverage of pericytes and reconstruction of new vascular basement membrane suggest that insulin is a potent accelerator of microvascular maturation, which may be involved in the mechanisms of insulin-induced wound healing.