Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A:2788-2795, 2013.