Intestinal disorders and resection caused by inherited or acquired abnormalities result in need of small intestine tissue transplantation in both children and adults. Tissue engineering offers a promising means to generate nascent intestinal tissue in vitro utilizing stem cells to provide tissue capable of transplantation. Serial perfusion and agitation with a hypotonic (10mM Tris-HCl), and a nonionic detergent (1% triton-X) with urea rinses was used to systematically create natural decellularized intestinal scaffolds. We attempted to recellularize the scaffolds with human fetal small intestine stem cells as a base for developing functional intestinal tissue. After five cycles of decellularization, histological examination and DNA analyses showed removal of cellular elements with preservation of the native architecture, connective tissue components and vasculature. Fetal small intestine cells attached to the decellularized SI matrix and could be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for SI regeneration. Maintenance of biomechanical, adhesion and angiogenic properties were also demonstrated. Fetal small intestine stem cells offer a unique and promising means to generate intestinal tissue for the purposes of modeling intestinal disease, understanding embryonic development and providing a source of material for therapeutic transplantation.