Abstract: Objective To explore the feasibility of differentiation of adipose stem cells into urothelial cells, evaluate the biocompatibility of cells and scaffolds, and provide experimental basis for further construction of tissue-engineered tissue. Methods The adipose derived stem cells(ADSCs) were isolated from the inguinal adipose tissue and bladder tissue of six 8-week-old male New Zealand white rabbits by collagenase digestion, and the urothelial cells were obtained by trypsin digestion. After flow cytometry identification, the third generation adipose stem cells and urothelial cells were co-cultured indirectly for 2 weeks, and the differentiated cells were phenotyped. Then the differentiated cells were implanted on the acellular matrix of bladder. The growth of the cells on the acellular matrix of bladder was observed by scanning electron microscopy and histological section. The surface antigen CD molecules were detected by flow cytometry. Results The adipose stem cells were cultured successfully and expanded in vitro. The cells expressed CD29(99.6%), CD44(98.2%), CD31(1.9%) and CD45(1.6%). After inducing differentiation by indirect co-culture, the expression of epithelial cell marker(UP1 a) was detected by immunofluorescence and Western blotting, but the uninduced adipose stem cells did not. Scanning electron microscopy(SEM) showed that the cells grew well and adhered well on the acellular matrix of bladder after induction. Histological examination showed that the cells on the surface of acellular matrix of bladder grew horizontally. Conclusion Induced adipose stem cells can differentiate into urothelial cells that can be used as seeding cells for urinary tissue engineering, which may be another choice out of urothelial cells. The bladder acellular matrix can support the good growth of the induced stem cells, which can be used as the carrier material for the repair and reconstruction of the urinary tissue engineering.