Diabetes-associated erectile dysfunction is associated with increased extracellular matrix deposition and reduced smooth muscle content in the corpus cavernosum. The mechanisms of these processes are not well understood. In this study, we investigated fibromuscular changes in the corpus cavernosum of rats with streptozotocin-induced diabetes to determine the mechanisms underlying pathologic changes in penile structure and function. Forty 8-week-old Sprague-Dawley rats were randomly distributed into control and diabetic groups. Diabetes was induced by a one-time intraperitoneal injection of streptozotocin 60 mg/kg. Twelve weeks later, erectile function was measured by cavernous nerve electrostimulation with real-time intracorporal pressure assessment. The penis was harvested for histologic examination (Masson trichrome stain, picrosirius red stain, Hart elastin stain, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and immunohistochemistry) and Western blot. Diabetes significantly attenuated erectile response to cavernous nerve electrostimulation. Diabetic animals exhibited a decreased smooth muscle/collagen ratio in the corpus cavernosum. The ratio of collagen I to II fibers was significantly lower in the corpora of diabetic rats compared with controls. Cavernous elastic fibers were fragmented in diabetic rats. There was up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway in diabetic rats. Phospho-Smad2 expression was higher in smooth muscle cells and fibroblasts of diabetic rats, as was the apoptotic index. The up-regulation of the transforming growth factor β1/Smad/connective tissue growth factor signaling pathway might play an important role in diabetes-induced fibrous-muscular structural changes and deterioration of erectile function.