Introduction The pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known. Aims The aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy-related ED. Methods Type 1 diabetes mellitus was induced in male rats by streptozotocin (75mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age-matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro-stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK-1, ROCK-2, phospho (P)-AKT (Ser473), and P-phosphatase and tensin homolog (P-PTEN) (Ser380/Thr382/383). Main Outcome Measures Effect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P-AKT/P-PTEN pathway in the MPG of diabetic rats. Results Erectile response was significantly (P<0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P<0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK-2 protein expressions in MPG were increased (P<0.05) and remained increased in hydroxyl fasudil-treated rats. P-AKT (Ser473) expression was decreased (P<0.05), whereas P-PTEN (Ser380/Thr382/383) expression was increased (P<0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P<0.05) in diabetic rats treated with hydroxyl fasudil. Conclusion Improved erectile function and restored P-AKT and P-PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED. Sezen SF, Lagoda G, Musicki B, and Burnett AL. Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: A role for neuronal ROCK. J Sex Med 2014;11:2164-2171.