Objective: To determine the feasibility and effectivity of allogenic frozen-thawed bladder mucosa for urethroplasty. Methods: Bladder mucosa was harvested from 6 New Zealand rabbits. Changes in the bladder mucosa as seen by histological and electron microscope examination were compared between the frozen-thawed and fresh groups. Twelve urethral stricture models were established and randomly divided into two groups. In the test group, we performed urethroplasty with allogenic frozen-thawed bladder mucosa, and the same operation was done in the control group, but using fresh bladder mucosa. The result of retrograde urethrography and histological changes of the urethral sample were compared postoperatively. Results: No obvious changes on histological and electron microscope examination were observed in the frozen-thawed bladder mucosa. Inflammation reaction of the surgical site in the test group was milder than that of the controls 2 weeks after surgery. The urethral epithelial cells grew well 2 weeks after surgery, but lots of epithelia were necrotic in the control group. The urethra of all rabbits in the test group had good continuity and the urethral lumen was large in the test group 2 months after surgery. There was a layer of urethral epithelium in the test group 2 months after surgery, whereas scar tissue was found in the control group. Conclusions: The freeze-thaw technique can maintain bladder mucosa structure and biological function. Frozen-thawed allogenic bladder mucosa may be a potential material for urethroplasty.