Diabetes mellitus-induced erectile dysfunction (DMED) has become a common disease in adult men that can seriously reduce the quality of life of patients, and new therapies are urgently needed. miRNA-100 has many targets and can induce autophagy and reduce fibrosis by inhibiting the mTOR pathway and the TGF-β pathway. However, no research has been conducted with miR-100 in the field of DMED, and the specific mechanism of action is still unclear.To ascertain the effects of miR-100 on corpus cavernosum tissue of DMED rats and vascular endothelial cells in a high glucose environment and to elucidate the relevant mechanisms in autophagy, fibrosis and inflammation to find a new approach for the DMED therapy.Thirty rats were divided into three groups: the control group, the DMED group, and the DMED + miR-100 group. Using intraperitoneal injections of streptozotocin, all rats except the control group were modeled with diabetes mellitus, which was verified using the apomorphine (APO) test. For rats in the DMED + miR-100 group, rno-miR-100-5p agomir (50 nmol/kg, every 2 days, 6 times in total) was injected via the tail vein. After 13 weeks, the erectile function of each rat was assessed using cavernous manometry, and the corpus cavernosum tissue was harvested for subsequent experiments. For cellular experiments, human coronary microartery endothelial cells (HCMEC) were divided into four groups: the control group, the high-glucose (HG, 40 mM) group, the HG + mimic group, and the HG + inhibitor group. The cells were cultured for 6 days and collected for subsequent experiments 2 days after transfection.Diabetic modeling impaired the erectile function in rats, and miR-100 reversed this effect. By measuring autophagy-related proteins such as mTOR/Raptor/Beclin1/p62/LC3B, we found that miR-100 could suppress the expression of mTOR and induce autophagy. The analysis of the eNOS/NO/cGMP axis function indicated that impaired endothelial function was improved by miR-100. By evaluating the TGF-β1/CTGF/Smad2/3 and NF-κB/TNF-α pathways, we found that miR-100 could lower the level of inflammation and fibrosis, which contributed to the improvement of the erectile function. Cellular experiments can be used as supporting evidence for these findings.MiR-100 can improve the erectile function by inhibiting mTOR and thus inducing autophagy, improving the endothelial function through the eNOS/NO/cGMP axis, and exerting antifibrotic and anti-inflammatory effects, which may provide new ideas and directions for the treatment of DMED.© 2024 American Society of Andrology and European Academy of Andrology.