Purpose: Several urethral conditions may require tissue substitution. One collagen-base biomaterial that recently emerged as an option is small intestinal submucosa (SIS). The aim of this study was to compare the results of SIS and buccal mucosa for urethral substitution in rabbits. Materials and Methods: Thirty-six North Folk male rabbits were randomized into three groups. In all animals, a 10 · 5 mm urethral segment was excised, and the urethral defect was repaired using a one-layer SIS patch (group I [GI]); four-layer SIS (group II [GII]); or buccal mucosa (group III [GIII]). Urethrography was performed preoperatively and after 12 weeks. After sacrifice, graft retraction was objectively measured using Scion Image Ò computer analysis and by calculation of ellipse area. The grade of fibrosis, inflammatory reaction, vascular/ epithelial regeneration, and collagen III/I ratio were analyzed by hematoxylin/eosin and Picrosirius red staining. Results: Urethrography confirmed a wide urethral caliber without any signs of strictures after surgery. Urethral fistulae was diagnosed in 8.3% of cases (1 animal each group). Average graft shrinkage was 55.2% in GI; 44.2% in GII; and 57.2% in GIII ( p < 0.05). The intensity of chronic inflammation, fibrosis, epithelium regeneration, and neovascularization was similar in all groups ( p > 0.05). Collagen III/I ratio was higher in GII (GI: 119.6; GII: 257.2 and GIII: 115.0); p < 0.01. Conclusions: The four-layer SIS is more advantageous than the one-layer SIS and buccal mucosa for urethral substitution in rabbits.