Background Diabetes mellitus (DM) induces impairment of male reproductive system and is considered as a key factor that could partially provide an explanation for male infertility. Thus, understanding the mechanism underlying DM-induced infertility will aid in the identification of novel therapeutic stratagems. Objectives To delineate the role of ROS/JAK2/NF kappa B pathway in DM-induced low reproductive function and impaired spermatogenesis. Additionally, to investigate the protective effect of monomeric Berberine (BB) that inhibits ROS/JAK2/NF kappa B pathway, in the pathogenesis of DM-induced infertility. Methods 12-week-old male Sprague-Dawley rats were divided into four groups: control group, DM group, control plus BB group, and DM plus BB group. Streptozotocin was used to induce DM. After treating the rats with BB for 4 weeks, fertility tests were conducted to investigate the reproductive function, and testis weight along with sperm motility was assessed through microscope. Oxidative stress was evaluated by DHE staining. TUNEL staining was utilized to detect the state of apoptosis. Cell experiments were carried out to define the role of BB in vitro. Immunohistochemistry, immunofluorescence, and Western blotting were employed to measure the protein expression. Results Our results indicate that the reproductive function of DM rats was low, accompanied by decreased testis weight and sperm motility in addition to the impairment of the seminiferous tubules. However, there was a significant improvement in the reproductive function parameters in the BB-treated DM rats. Subsequently, our data revealed that DM rats produce an increased level of ROS in the testis, which activates JAK2 further activating the NF kappa B pathway, leading to increased apoptosis and impaired cells in the testicles. However, BB could attenuate the ROS production and abrogate activation of JAK2/NF kappa B pathway, thus inhibiting the apoptosis in the testicular cells of DM rats. Conclusion ROS/JAK2/NF kappa B pathway is involved in the DM-induced low reproductive function and impaired spermatogenesis. BB can play a protective role in preserving the reproductive function and spermatogenesis in DM by inhibiting ROS/JAK2/NF kappa B pathway.