We developed a stromal cell-derived factor-1 alpha (SDF-1 alpha)-aligned silk fibroin (SF)/three-dimensional porous bladder acellular matrix graft (3D-BAMG) composite scaffold for long-section ventral urethral regeneration and repair in vivo. SDF-1 alpha-aligned SF microfiber/3D-BAMG, aligned SF microfiber/3D-BAMG, and nonaligned SF microfiber/3D-BAMG scaffolds were prepared using electrostatic spinning and wet processing. Adipose-derived stem cell (ADSC) and bone marrow stromal cell (BMSC) migration was assessed in the SDF-1 alpha-loaded scaffolds. Sustained SDF-1 alpha release in vitro and vivo was analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting, respectively. The scaffolds were used to repair a 1.5 x 1 cm(2) ventral urethral defect in male rabbits in vivo. General observation and retrograde urinary tract contrast assessment were used to examine urethral lumen patency and continuity at 1 and 3 months post-surgery. Postoperative rehabilitation was evaluated using histological detection. The composite scaffolds sustained SDF-1 alpha release for over 16 days in vitro. SDF-1 alpha-aligned SF nanofiber promoted regeneration of urethral mucosa, submucosal smooth muscles, and microvasculature, increased cellular proliferation, and reduced collagen deposition. SDF-1 alpha expression was increased in reconstructed urethra at 3 months post-surgery in SDF-1 alpha-aligned SF group. SDF-1 alpha-aligned SF microfiber/3D-BAMG scaffolds may be used to repair and reconstruct long urethral defects because they accelerate urethral regeneration.