Pharmacological therapy of diabetes mellitus-induced erectile dysfunction (DMED) is intractable owig to the poor response to phosphodiesterase type 5 inhibitors (PDE5i). The surge in the number of diabetic patients makes it extremely urgent to find a novel therapy for DMED. Ferroptosis is a recently discovered form of cell death evoked by lipid peroxidation and is related to several diabetic complications. GPX4, an important phospholipid hydroperoxidase, can alleviate ferroptosis and maintain redox balance via reducing lipid peroxides. However, whether GPX4 can be a prospective target of DMED needs to be determined. Fifty rats were randomly divided into control group, DMED group, DMED + negative control group (DMED + NC group), DMED + low-dose group (1 x 10(6) infectious units), and DMED + high-dose group (2 x 10(6) infectious units). Erectile function was assessed 4 weeks after intracavernous injection of GPX4 or negative control lentivirus. The penile shafts were collected for subsequent molecular biological and histological analysis. The results demonstrated that erectile function of the rats in DMED and DMED + NC groups was extremely impaired and was improved in a dose-dependent manner with GPX4 lentivirus (GPX4-LV) injection. Additionally, upregulation of the ACSL4-LPCAT3-LOX pathway, iron overload, oxidative stress, fibrosis, and decreased endothelial and smooth muscle cell numbers were observed in the corpus cavernosum of DMED group. Meanwhile, the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway was inhibited, and the Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway was promoted in DMED rats. The above histologic alterations and related molecular changes were alleviated after GPX4-LV injection. The results revealed that GPX4 improved erectile function by modulating ferroptosis during DMED progression. This finding is of paramount significance in deciphering the molecular mechanism of hyperglycemia-induced ferroptosis, thereby providing a prospective target for preventing the development of DMED.