To assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model. Autologous ADSCs were isolated, expanded and identified by flow cytometry. In the experimental group, ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits. Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits. Cystography was performed at 4, 12 and 24 weeks after grafts implantation. Following cystography, the animals were killed and grafts were harvested; H&E and immunohistochemical staining were performed with cytokeratin AE1/AE3, smooth muscle alpha-actin and S-100 markers. Flow cytometry demonstrated that the ADSCs expressed CD90, CD44, CD105, CD166 and CD34, but not CD45 or CD106. The cells demonstrated good biocompatibility with BAMGs. At 24 weeks, in the experimental group, the reconstructed bladders reached a mean volume of 94.68 +/- 3.31% of the pre-cystectomy bladder capacity. Complete regeneration of smooth muscle and nerve tissue was evident. Regenerated SMCs, urothelium and nerve cells stained positively for alpha-smooth muscle actin, AE1/AE3 and S-100. In the control group, the mean bladder volume was 69.33 +/- 5.05% of the pre-cystectomy volume; histologically, the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue. These data demonstrate that seeding ADSCs onto BAMGs promote regeneration of smooth muscle and nervous tissue regeneration in a rabbit model. This compound graft was more suitable for bladder reconstruction than BAMG alone.