Diabetes mellitus causes impaired wound healing. In this study the potential wounds healing activities of topical chitosan/ Zinc oxide nanocomposite membrane and local insulin injection in diabetic rats were evaluated. Diabetes was induced by single IP injection of Streptozotocin (STZ) at a dose of 50mg/kg b.wt. Forty eight male rats divided into Six groups. Group I: control wounded, non-treated , non-diabetic rats, Group II : wounded diabetic non treated, Group III : Normal Wound rats treated with chitosan/ Zinc oxide nanocomposite membrane, Group IV:Diabetic wounded treated with chitosan/ Zinc oxide nanocomposite membrane rats, Group V: wounded diabetic rats treated with local insulin injection, Group VI : Wounded diabetic rats treated with chitosan/ Zinc oxide nanocomposite membrane and local insulin injection. After 14 days of wound treatment, rats were euthanized and the skin tissue were collected for (EGF), (PDGF), (MMP9) and miRNA 21 gene expression analysis. A significant down-regulation of EGF, PDGF and miRNA 21 with up-regulation in MMP 9 were observed in diabetic non treated wound as compared with control wounded. Meanwhile, a significant increase of EGF, PDGF and miRNA 21 with decrease in MMP 9 gene expression were observed in insulin, Chitosan/ZnO membrane treatment alone or in combination in wounded diabetic rats. Conversely, MMP9 was a significantly down regulated after different treatment. The finding indicated that topical Chitosan/ZnO nanocomposite membrane and insulin injection exhibited great effect on acceleration of diabetic wound healing via increasing pro-angiogenic effect, re-epithelialization and remodeling of ECM.