Abstract: Objective To investigate the feasibility of autologous bone marrow mesenchymal stem cells (BMSCs) seeded on bladder acellular matrix graft (BAMG) for bladder reconstruction in a canine model. Methods This study included 25 mongrel dogs. Five dogs were sacrificed for the preparation of BAMG. Twenty dogs were randomly divided into two groups and received partial cystectomy. In group A, the bladder defect was repaired with unseeded BAMG. In group B, the bladder defect was repaired with autologous BMSCs-seeded BAMG. The bladders were retrieved and studied histologically and immunohistochemically at the time point of 1, 2, 4, 8, 12 weeks after surgery to evaluate tissue regeneration. Results All dogs survived the procedure. Histopathological examination in group B showed there was urothelium developed at the end of the 2 weeks. By 8 and 12 weeks all bladder wall components were regenerated in the repaired area, which were similar to normal bladder tissue. In group A, urothelium regeneration was observed at the end of the 4 weeks, whereas smooth muscle was still not well-formed by 12 weeks. Conclusion Autologous BMSCs-seeded BAMG could promote the repair of bladder defect, which is superior to unseeded BAMG in regenerative properties.