Colon decellularization provides three-dimensional biologic scaffold without any cell elements with preservation of extracellular matrix in order to enable autologous cell seeding for tissue augmentation without any immunological response. This study was performed to investigate the safety and feasibility of sheep colon decellularization as a first step of colon tissue engineering. The process of sheep colon decellularization was done in four stages which included scaffold preparation, histologic examination and microscopic investigations to reveal the remaining cellular deposits, biomechanical evaluation and collagen quantification studies by measurement of hydroxyproline content of normal and decellularized sheep colon. Decellularized colon scaffold revealed complete cell removal under a light microscope while 4',6-diamidino-2-phenylindole, di-hydrochloride (DAPI) staining confirmed no deoxyribonucleic acid (DNA) residues. Decellularized colon displayed preserved ultrastructure, comparable biophysical properties (resistance to unidirectional stretch forces) and higher hydroxyproline content. The results of biomechanical tests proved that the decellularized matrix did not bear any unexpected damages or structural changes which would make it unable to tolerate in vivo forces and stretches. The microscopic images captured after staining the tissue with Picro-sirius red also showed that the collagen in extracellular matrix is well preserved; this was confirmed by scanning electron microscopy. This implies that the scaffold prepared by this method is suitable for tissue augmentation or transplantation. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.