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Background: Diabetic chronic wounds are the most critical clinical issues. Although a wide range of wound dressings have been designed, few of them act sufficiently in repairing and protecting wounds. Aim and Objectives: This study aimed to investigate the burn wound healing effect of neonatal fibroblasts seeded into chitosan hydrogel in diabetic rats. Material and Methods: Two burn wounds were created in male Wistar diabetic rats. The rats were divided into groups treated with fibroblast cells, chitosan hydrogel, fibroblast cell cultured chitosan hydrogel (chitosan + fibroblasts group), and control untreated group. Macroscopic and microscopic examination, wound area measurement, measurement of skin density and thickness, and also skin elasticity were applied to evaluate wound healing. The data were analyzed using ANOVA. Results: Treatment of diabetic burn wound with chitosan + fibroblasts accelerated healing process by promoting re-epithelialization and reducing inflammatory responses in skin tissue. Skin elasticity, thickness and density were higher in chitosan + fibroblasts group compared with control group 3 weeks after treatment. Conclusion: The results of the present study show that dressing the diabetic burn wound with neonatal fibroblasts seeded into chitosan scaffold plays a fundamental role in wound healing process.