Transforming growth factor (TGF) β1 has been suggested to have an important role in cavernous fibrosis and resultant erectile dysfunction. For further elucidation of TGFβ1 signaling in association with cavernous fibrosis, we developed a rat model of cavernous fibrosis using TGFβ1-producing NIH 3T3 fibroblasts (NIH 3T3-TGFβ1). The NIH 3T3-TGFβ1 cells were injected into male Sprague-Dawley rats intracavernously. Masson trichrome staining at 20 days postinjection showed multiple fibrous scars in the rats injected with the NIH 3T3-TGFβ1 cells (group 3), whereas no histological evidence of cavernous fibrosis was found in the control rats (group 1) or the recombinant human TGFβ1 protein-injected rats (group 2). Immunohistochemical staining revealed a higher expression of TGFβ1 and its type II receptor in group 3 than in groups 1 and 2. Electrostimulation of the cavernous nerve revealed that the maximal intracavernous pressure was significantly lower in group 3 than in groups 1 and 2 (P < 0.01). The expression of transgenic TGFβ1 mRNA continued to 10 days after injection of the cells. The NIH 3T3-TGFβ1 cells sufficiently induced relatively long-lasting cavernous fibrosis. This novel animal model may contribute to future investigations of the pathogenesis of penile fibrosis associated with TGFβ1 signaling and the development of new therapeutics targeting this pathway.