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In this study primary mouse embryonic fibroblasts (p-MEF) were cultured and seeded on bubaline acellular diaphragm matrix (ADM) to evaluate its healing potential on full thickness skin wounds. A 20 x 20 mm2 sized full thickness skin defect was created on dorsum of forty eight Sprague Dawley rats and randomly divided into four equal groups. The defect in group I (control) was left open whereas, groups II, III and IV were covered with autograft, ADM and p-MEF seeded ADM, respectively. Wound healing was evaluated on the basis of gross, immunologic and histopathologic observations. Gross healing observation with p-MEF seeded ADM group showed significantly (P < 0.05) higher wound contraction as compared to others. There was higher B-cell response in animals with ADM and p-MEF seeded ADM implants. Stimulation index of spleenocytes was higher with p-MEF seeded ADM as compared to ADM by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The p-MEF seeded ADM implantation caused markedly fast wound closure with well-formed granulation tissue dominated by fibroblast proliferation, collagen deposition, and complete early regenerated epithelial layer. On critical analysis of the observed parameters it was found that p-MEF seeded ADM has healing potential and shown better healing of full thickness skin wounds in rats.