This study was undertaken to establish a Peyronie's disease model with penile curvature by using recombinant transforming growth factor-β1 (TGF-β1) protein or adenovirus (ad-TGF-β1). Four-month-old male Sprague–Dawley rats were divided into seven groups (n = 18 per group): G1 received a single injection of saline into the tunica albuginea (0.1 mL); G2, repeated injections of ad-LacZ (days 0, 3, and 6; 1 × 1010 particles/0.1 mL respectively); G3, a single injection of recombinant TGF-β1 protein (700 ng/0.1 mL); G4, repeated injections of recombinant TGF-β1 protein (days 0, 3 and 6; 700 ng/0.1 mL respectively); G5, a single injection of low-dose ad-TGF-β1 (1 × 1010 particles/0.1 mL); G6, a single injection of high-dose ad-TGF-β1 (3 × 1010 particles/0.1 mL); and G7, repeated injections of low-dose ad-TGF-β1 (days 0, 3, and 6; 1 × 1010 particles/0.1 mL respectively). Penile curvature was evaluated 30, 45 and 60 days after treatment, and the penis was then harvested for histological examination. Repeated injection of low-dose ad-TGF-β1 not only induced fibrous scar in the tunica, which lasted up to 60 days after injection, but also resulted in significant penile curvature by artificial erection test 45 days after treatment. A peculiar histological finding in this group was trapping of inflammatory cells in the tunica, subsequent fibrosis, and formation of cartilage and calcification as well as loss of elastin fibres. This model involving repeated injection of ad-TGF-β1 may contribute to further investigation of the pathogenesis of Peyronie's disease and the development of new therapeutics targeting this pathway.