In a previously reported attempt to regenerate small intestine with autologous tissues, collagen scaffolds were used without cell seeding or with autologous mesenchymal stem cell seeding. However the regenerated intestine lacked a smooth muscle layer. To accomplish regeneration of a smooth muscle layer, this present study used collagen scaffolds seeded with the smooth muscle cells (SMC) in a canine model. Autologous SMC were isolated from stomach wall and cultured. Two types of scaffolds were fabricated: in SMC (+), cultured SMCs were mixed with collagen solution and poured into a collagen sponge; and in SMC (-), SMCs were omitted. Both scaffolds were implanted into defects of isolated ileum as a patch graft. Animals were euthanized at 4, 8, and 12 weeks; for the last time point, the ileal loop had been reanastomosed at 8 weeks. At 12 weeks, the SMC (-) group showed a luminal surface covered by a regenerated epithelial cell layer with very short villi; however only a thin smooth muscle layer was observed, representing the muscularis mucosae. In the SMC (+) group, the luminal surface was covered completely by a relatively well-developed epithelial layer with numerous villi. Implanted SMCs were seen in the lamina propria and formed a smooth muscle layer. Thus, we concluded that collagen sponge scaffolds seeded with autologous SMCs have a potential for small intestine regeneration.