Background: Oxidative stress is a significant contributor to the poor treatment efficacy on erectile dysfunction induced by diabetes mellitus (DMED). Thus, understanding the mechanism underlying oxidative stress will aid in the identification of novel therapeutic targets. Aim: To define the role of Janus kinase 2 (JAK2) in mediating oxidative stress in the corpus cavernosum smooth muscle cells (CCSMCs) and to investigate the therapeutic effect of monomeric berberine (BB), which inhibits JAK2, in the pathogenesis of DMED. Methods: Streptozotocin was used to establish type I diabetic rat models and apomorphine tests were conducted to determine DMED rats. Eighteen DMED rats were divided into the DMED group and the DMEDthornBB group, whereas another 10 age-matched rats formed the control group. CCSMCs were isolated from the corpus cavernosum of rats and were treated with the JAK2 inhibitor alpha cyanano-(3,4-hydroxyl)N-benzophenylamine (AG490) and/or BB. Outcomes: Metabolic parameters; erectile function; histologic and molecular alterations. Results: Erectile function was impaired and excessive oxidative stress was found in the DMED group. Excessive oxidative stress led to decreased expression level of phosphorylated endothelial nitric oxide synthase at serine 1177/endothelial nitric oxide synthase and increased expression level of Ras homolog gene family and Rho kinase 1/2. Meanwhile, the relative expression ratio of phosphorylated JAK2/JAK2 was significantly greater in the DMED group than that in the other groups. In vitro, oxidative stress was significantly reduced along with reduced intracellular calcium upon treatment with the JAK2 inhibitor, AG490. Moreover, the CCSMCs treated with BB showed changes similar to those upon treatment with AG490. In vivo experiments also confirmed that the erectile function of the DMEDthornBB group was improved, accompanied by decreased phosphorylated JAK2/ JAK2 and decreased oxidative stress. Clinical Translation: JAK2 can be used as a therapeutic target and BB can be used as a potential drug for the clinical treatment of DMED. Strengths and Limitations: This study examines the promoting effect of JAK2 on oxidative stress occurrence in the corpus cavernosum and on the development of DMED in both animal experiments and cell experiments, as well evaluates the inhibitory effect of BB on JAK2 and its therapeutic effect on DMED. The main limitation of our current study is the lack of an appropriate means for activating JAK2. Conclusions: JAK2 can induce DMED by enhancing oxidative stress and BB can play a role in treating DMED by inhibiting JAK2 and reducing oxidative stress. Our study provides an option and an idea for further studies on the pathogenesis and treatment of DMED. Copyright (C) 2019, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.