Introduction Bilateral cavernous nerve injury (BCNI) causes profound penile changes such as apoptosis and fibrosis leading to erectile dysfunction (ED). Histone deacetylase (HDAC) has been implicated in chronic fibrotic diseases. Aims This study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI-induced ED and penile fibrosis. Methods Five groups of rats (8-10 weeks, n=10/group) were utilized: (i) sham; (ii and iii) BCNI 14 and 30 days following injury; and (iv and v) BCNI treated with HDAC inhibitor valproic acid (VPA 250mg/kg; 14 and 30 days). All groups underwent cavernous nerve stimulation (CNS) to determine intracavernosal pressure (ICP). Penile HDAC3, HDAC4, fibronectin, and transforming growth factor-1 (TGF-1) protein expression (Western blot) were assessed. Trichrome staining and the fractional area of fibrosis were determined in penes from each group. Cavernous smooth muscle content was assessed by immunofluorescence to alpha smooth muscle actin (-SMA) antibodies. Main Outcome Measures We measured ICP; HDAC3, HDAC4, fibronectin, and TGF-1 protein expression; penile fibrosis; penile -SMA content. Results There was a voltage-dependent decline (P<0.05) in ICP to CNS 14 and 30 days after BCNI. Penile HDAC3, HDAC4, and fibronectin were significantly increased (P<0.05) 14 days after BCNI. There was a slight increase in TGF-1 protein expression after BCNI. Histological analysis showed increased (P<0.05) corporal fibrosis after BCNI at both time points. VPA treatment decreased (P<0.05) penile HDAC3, HDAC4, and fibronectin protein expression as well as corporal fibrosis. There was no change in penile -SMA between all groups. Furthermore, VPA-treated BCNI rats had improved erectile responses to CNS (P<0.05). Conclusion HDAC-induced pathological signaling in response to BCNI contributes to penile vascular dysfunction. Pharmacological inhibition of HDAC prevents penile fibrosis, normalizes fibronectin expression, and preserves erectile function. The HDAC pathway may represent a suitable target in preventing the progression of ED occurring post-radical prostatectomy. Hannan JL, Kutlu O, Stopak BL, Liu X, Castiglione F, Hedlund P, Burnett AL, and Bivalacqua TJ. Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats. J Sex Med 2014;11:1442-1451.