The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N = 10), 3D porous BC alone (group 2, N = 10), and 3D porous BC seeded with lingual keratinocytes (group 3, N = 10) were used to repair rabbit ventral urethral defects (2.0 × 0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23 ± 1.14 μm and 67.00 ± 6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p < 0.05). Comparisons of smooth muscle content and endothelia density among the three groups revealed a significant increase at each time point (p < 0.05). Our results demonstrated that 3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction.