Background: Tissue engineered urethral substitutes could offer alternative approaches for urethral regeneration. In this work, we generated and evaluated in vivo a multilayered urethral substitute generated with nanostructured fibrin-agarose biomaterials. Methods: A three-layered urethral substitute was generated and grafted to repair surgically induced penile urethra defects in rabbits (acellular multilayered urethral substitute graft (UREG)). Native urethras served as positive controls (positive control (P-CTR)), unrepaired defects as negative controls (negative control (N-CTR)), and oral mucosa autograft (AUTO), as the gold standard treatment. Results were analyzed after 3 months for surgical feasibility, complications, urine stain patterns and histological, histochemical and immunohistochemical analyses. Results: All procedures were feasible, with no severe complications. Both in N-CTR and AUTO groups, 2/5 animals developed urethral fistulas, with none in UREG. Altered urine stains appeared in 3/5 of N-CTR and AUTO animals, and in 1/5 of UREG. Histologically, the UREG implants resembled native urethra, with high expression of pancytokeratin, cytokeratins 7, 8 and 19, involucrin and uroplakin, and stromal expression of versican, biglycan and decorin, with similar collagen and glycosaminoglycan contents. However, UREG grafts showed lower alcian blue staining intensity, higher number of blood vessels and a discontinuous muscular layer at the grafting site. In contrast, N-CTR and AUTO groups failed to reproduce the structure and molecular composition of the native urethra. Conclusions: A multilayered fibrin-agarose urethral substitute generated by tissue engineering could support urethral regeneration in an animal model of urethral repair. These findings support its potential as a promising alternative for urethral reconstruction.