In some types of tracheal disease, tracheal resection is required. For patients with tracheal resection, artificial grafts, made from collagen sponge with a spiral polypropylene stent and mesh, have been clinically used by our group. However, epithelial regeneration was confirmed to be slow. In the present study, we investigated the potential of gingival fibroblasts (GFBs) and adipose-derived stem cells (ASCs) as autologous transplanted cells in combination with artificial graft for tracheal epithelial regeneration. In in vitro co-culturing with tracheal epithelial cells, GFBs stimulated epithelial cell differentiation and reconstruction of a pseudostratified epithelium. ASCs stimulated epithelial cell proliferation and reconstruction of a multi-layered epithelium. Subsequently, we prepared three kinds of bioengineered scaffolds from GFBs and/or ASCs and implanted them into rat tracheal defects. The bioengineered scaffolds containing GFBs were covered with tracheal epithelial cells after 1 week, and highly ciliated epithelium was formed after 2 weeks of transplantation. The bioengineered scaffold containing ASCs induced thick epithelium, and then pseudostratified epithelium containing goblet cells was formed. Furthermore, the application of both GFBs and ASCs had synergistic effects on tracheal epithelial regeneration, suggesting that bioengineered scaffolds containing GFBs and ASCs are useful for hastening tracheal epithelial regeneration.