Introduction: We have used a long-acting nitric oxide (NO)-releasing polymer to develop injectable biodegradable microspheres capable of localized NO release over prolonged periods of time. Aim: The aim of this study was to evaluate the therapeutic potential of these microspheres for diabetes-related erectile dysfunction (ED) in the rat model. Methods: NO-releasing microspheres were incubated in physiologic buffer, and in vitroNO release was measured using a Griess assay. To ensure no migration, microspheres were fluorescently tagged and injected into the corpus cavernosum of adult rats, and fluorescent imaging was performed weekly for 4 weeks, at which point rats were sacrificed. To assess physiologic efficacy, diabetes was induced in 40 rats using streptozotocin (STZ), whereas 10 rats were kept as age-matched controls. Diabetic rats were divided into four groups: no treatment, sildenafil, NO-releasing microspheres, and combination therapy. For each rat, the cavernosal nerve (CN) was stimulated at various voltages, and intracavernosal pressure (ICP) and mean arterial pressure (MAP) were measured via corpus cavernosum and carotid artery catheterization, respectively. Long-term efficacy was determined by injecting diabetic rats with microspheres and measuring erectile response at predetermined intervals for up to 5 weeks. Main Outcome Measures: Erectile response was determined via calculation of mean peak ICP/MAP and area under curve (AUC) for each experimental group. Results: Under physiologic conditions in vitro, microspheres continued NO release for up to 4 weeks. Fluorescent imaging revealed no detectable signal in tissues besides cavernosal tissue at 4 weeks postinjection. Upon CN stimulation, peak ICP/MAP ratio and AUC of diabetic rats improved significantly (P<0.05) in microsphere and combination therapy groups compared with no treatment and sildenafil groups. In long-term efficacy studies, microspheres augmented the effect of sildenafil for 3 weeks following injection (P<0.05). Conclusions: NO-releasing microspheres significantly improved erectile response in diabetic rats for 3 weeks and hence offer a promising approach to ED therapy, either as monotherapy or combination therapy. © 2013 International Society for Sexual Medicine.