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Reconstructive surgery for urethral defects employing tissue‐engineered scaffolds represents an alternative treatment for urethroplasty. The aim of this study was to compare the therapeutic efficacy of the bilayer poly‐D,L‐lactide/poly‐ε‐caprolactone (PL‐PC) scaffold seeded with allogenic mesenchymal stem cells (MSCs) for urethra reconstruction in a rabbit model with conventional urethroplasty employing an autologous buccal mucosa graft (BG). The inner layer of the scaffold based on poly‐D,L‐lactic acid (PL) was seeded with MSCs, while the outer layer, prepared from poly‐ε‐caprolactone, protected the surrounding tissues from urine. To track the MSCs in vivo, the latter were labeled with superparamagnetic iron oxide nanoparticles. In rabbits, a dorsal penile defect was reconstructed employing a BG or a PL‐PC graft seeded with nanoparticle‐labeled MSCs. In the 12‐week follow‐up period, no complications were detected. Subsequent histological analysis demonstrated biointegration of the PL‐PC graft with surrounding urethral tissues. Less fibrosis and inflammatory cell infiltration were observed in the experimental group as compared with the BG group. Nanoparticle‐labeled MSCs were detected in the urothelium and muscular layer, co‐localizing with the urothelium cytokeratin marker AE1/AE3, indicating the possibility of MSC differentiation into neo‐urothelium. Our results suggest that a bilayer MSCs‐seeded scaffold could be efficiently employed for urethroplasty. [ABSTRACT FROM AUTHOR]