Conventional tracheal reconstruction techniques are not successful at restoring functional units in situations with extensive damage involving more than half the length of the trachea. For the first time, we investigated in vivo tissue-engineered trachea regeneration from a decellularized cadaveric trachea matrix with seeded adult adipose tissue-derived mesenchymal stem cells (MSCs) and investigated the integration of the matrix into the recipient tracheal side. For the procedure, 1.8-cm grafts were prepared from 3.5-cm tracheas of three donor rabbits. Then, tracheal grafts were rendered nonimmunogenic using a decellularization technique. MSCs isolated from recipient rabbit adipose tissue were cultured and marked before being seeded in the decellularized matrix. A total of 1.8 cm of the recipient tracheas was replaced with either a decellularized tracheal matrix (group 1) or tracheal matrix-seeded MSCs (group 2). Rabbits survived 17 ± 2 days in the first group, and the causes of death were separation in the anastomosis region, airway obstruction, and infection. In the second group, animals were sacrificed on the 30th, 60th, and 90th days of follow-up. Histopathological analysis revealed the integration of MSCs seeded-decellularized cadaveric tracheas to the recipient tracheal sides and increased angiogenesis. The MSCs were traced by fluorescence microscopy in the ciliated epithelium, under the epithelium, and in the cartilage of the integrated new trachea. Tracheas generated by autologous cells and tissue-engineering techniques will be a great source for the treatment of life-threatening tracheal injuries after the completion of related studies.