Tissue engineering using a biocompatible scaffold with various cells might be a solution for tracheal reconstruction. We investigated the plausibility of using mesenchymal stem cells (MSCs) seeded on a porcine cartilage powder (PCP) scaffold for tracheal defect repair. PCP made with minced and decellularized porcine articular cartilage was molded into a 5 × 12 mm (height × diameter) scaffold. MSCs from young rabbit bone marrow were expanded and cultured with the PCP scaffold. After 7 weeks culture, the tracheal implants were transplanted on a 5 × 10 mm tracheal defect in six rabbits. 6 and 10 weeks postoperatively, the implanted area was evaluated. None of the six rabbits showed any sign of respiratory distress. Endoscopic examination revealed that respiratory epithelium completely covered the regenerated trachea and there were no signs of collapse or blockage. A patent luminal contour of the trachea was observed on the computed tomography scan in all six rabbits and the reconstructed areas were not narrow compared to normal adjacent trachea. Histologic examination showed that neo-cartilage was successfully produced with minimal inflammation or granulation tissue. Ciliary beating frequency of the regenerated epithelium was not significantly different from the normal adjacent mucosa. MSCs cultured with a PCP scaffold successfully restored not only the shape but also the function of the trachea without any graft rejection.