Urethral reconstruction remains a challenge. Indeed, the use of oral mucosa, the reference biomaterial for urethroplasty, is associated with two main drawbacks: the limited availability of autologous tissues and potential short- and long-term complications, especially for patients with recurrences or severe anomalies. Therefore, the development of alternative approaches, such as urethral tissue engineering, is necessary. A new type of human urethral substitute devoid of exogenous biomaterials has been reconstructed in vitro. It presented sufficient mechanical strength and had histological and functional properties comparable to native tissues. These reconstructed tissues were implanted in vivo to repair hypospadias induced in tacrolimus-immunosuppressed rabbits via a two-stage urethroplasty. In the first stage, the distal part of the native urethra was removed, and a flat graft was implanted, leaving the urethra open proximally. Twelve weeks later, the graft was tubularized to create a neourethra, reproducing the usual clinical scenario. The results obtained for the experimental group were less effective than for the control group, with a success rate of 50% after excluding the animal affected by unwanted events unrelated to urethroplasty, and it is possible that the animal model or surgical technique used was not suitable and should be modified. Nevertheless, half of the urethral substitutes grafted on rabbits showed successful integration. These self-assembled artificial tissues represent promising substitutes for urethroplasty.