BACKGROUND: Acellular bladder submucosa is a natural extracellular matrix, which is mainly composed of collagen Ⅰ and Ⅲ. It is regarded as an ideal biological scaffold material. OBJECTIVE: To evaluate the biocompatibility of acellular bladder submucosa as a tissue engineered scaffold material. METHODS: Pig urinary bladder was immersed in the solution of PBS and sodium azide for a night, and the mucosa was removed. Acellular bladder submucosa was prepared using continuous hypotension, freeze-thawed treatment and NaOH spallation. The biocompatibility of acellular bladder submucosa was evaluated through histologic structure, DNA residual, cytotoxicity, cell adhesion, as well as subcutaneous inflammatory reactions. RESULTS AND CONCLUSION: The cell components were completely eliminated after deoellularization treatment, while the extracellular matrix was remained intact as normal bladder:According to MTT results, cytotoxicity of acellular bladder matrix was assigned to be the first grade. No DNA signal was observed after extraction, and the matrix also supported porcine smooth muscle cell attachment and proliferation. Subcutaneous implantation of the matrix indicated that the acellular bladder submucosa trigger no immunologic rejection reaction obviously. The results demonstrated that: the acellular bladder submucosa prepared here exhibits excellent biocompatibility, which can be used as substitution in tissue-engineering field.