The preparation method of urethral extracellular matrix (ECM) was introduced and the biocompatibility of urethral ECM was studied. The urethral segmental defect was created artificially in animal models, which were followed by reconstruction with prepared urethral ECM. And postoperative histologic and pathologic exams were employed to evaluate the biocompatibility of urethral ECM. The experimental results show that urethral ECM was biocompatible and can promote cellular interaction as well as tissue development. Urethrography and urodynamic evaluation revealed that there was no difference between the normal and new tissue. The urethral ECM appears to be a useful material for urethral repair in the rabbit. It is biocompatible and for tissue handling.