Abstract Background Congenital or acquired abnormalities may result in ureteral malformation, trauma, or defect. Traditional reconstructive methods are often associated with numerous complications. Tissue engineering technology may provide an alternate avenue for ureteral reconstruction. In this study, we constructed tissue-engineered tubularized grafts (TETGs) by seeding homologous adipose-derived stem cells (ADSCs) and bladder smooth muscle cells (SMCs) into bladder submucosa matrix (BSM) for ureteral reconstruction in rabbit models. Methods ADSCs and bladder SMCs were seeded onto 2 sides of the BSM, respectively. Then the grafts were used to construct TETGs of 4.0 cm length and 8.0 mm diameter and were transplanted into the omentum of rabbits for 2 weeks before ureteral reconstruction. The 4.0-cm segment of the ureter was replaced by the TETG. Evolutionary formation of tissue structures and degree of epithelization were evaluated with the use of histologic and immunohistochemical techniques at 2, 4, 8, and 16 weeks after implantation. Results All of the rabbits were alive until they were killed. Histologic and immunohistochemical analyses showed consistent regeneration of mature and functional urothelium. At 16 weeks after TETG implantation, multilayered urothelium covered the entire lumen, with visible neovascularization in the center and formation of organized smooth muscle bundles. Conclusions We successfully constructed a tissue-engineered transplanted graft by seeding ADSCs and SMCs onto the BSM for ureteral repair and reconstruction in a rabbit model.