For successful tracheal reconstruction, tissue-engineered artificial trachea should meet several requirements, such as biocompatible constructs comparable to natural trachea, coverage with ciliated respiratory mucosa, and adequate cartilage remodeling to support a cylindrical structure. Here, we designed an artificial trachea with mechanical properties similar to the native trachea that can enhance the regeneration of tracheal mucosa and cartilage through the optimal combination of a two-layered tubular scaffold and human induced pluripotent stem cell (iPSC)-derived cells. The framework of the artificial trachea was fabricated with electrospun polycaprolactone (PCL) nanofibers (inner) and 3D-printed PCL microfibers (outer). Also, human bronchial epithelial cells (hBECs), iPSC-derived mesenchymal stem cells (iPSC-MSCs), and iPSC-derived chondrocytes (iPSC-Chds) were used to maximize the regeneration of tracheal mucosa and cartilage in vivo. After 2 days of cultivation using a bioreactor system, tissue-engineered artificial tracheas were transplanted into a segmental trachea defect (1.5-cm length) rabbit model. Endoscopy did not reveal granulation ingrowth into tracheal lumen. Alcian blue staining clearly showed the formation of ciliated columnar epithelium in iPSC-MSC groups. In addition, micro-CT analysis showed that iPSC-Chd groups were effective in forming neocartilage at defect sites. Therefore, this study describes a promising approach for long-term functional reconstruction of a segmental tracheal defect.