Objective To investigate the biocompatibility of acellular urinary bladder submucosa (AUBS). Methods The acellular collagen matrix of human urinary bladder submucosa was developed using freeze-thawed enzymatic treatment and freeze-drying technique. Human oral keratinocytes were cultured and seeded on AUBS at a density of 2×106/ml in vitro.The proliferation of the cells were observed. Pockets were created in the abdominal muscle wall of 18 SD rats. AUBS in size 1 cm×1 cm was implanted into the pocket. The grafts were observed by light microscope 3, 6, 10, 14, 21 and 28 days after operation. Results AUBSmainly consisted of collagen fibers with a three-dimensional network structure. After the oral keratinocytes were seeded, continous oral epithelium layer was formed on the surface of AUBS after 10 days in vitro. Histological observation of the grafted AUBS showed progressive cell infiltration at 6 days. New capillaries formed at 14 days. The collagen fibers arranged regularly at 28 days after implantation. Conclusion Freeze-dried AUBS may be used as a suitable scaffold for tissue regeneration, which can induce cell proliferation both in vivo and in vitro and has good biocompatibilty.