Prolidase is a cytosolic exopeptidase that cleaves iminodipeptides with carboxy-terminal proline or hydroxyproline and plays major role in collagen turnover. Collagen is the essential content playing a key role in the stability/instability of atherosclerotic plaque and progression of endothelial dysfunction that may have a role in the pathogenesis of erectile dysfunction (ED). Consequently, in this study we sought to determine serum prolidase activity and markers of oxidative stress such as lipid hydroperoxide and total free sulfhydryl in vasculogenic ED. We have evaluated 92 patients with vasculogenic ED and 50 control cases with clinical and laboratory investigation. We have measured serum prolidase activity and serum total free sulfhydryl levels spectrophotometrically. Serum lipid hydroperoxide levels were determined with ferrous ion oxidation-xylenol orange method. We assessed the association of serum prolidase activity with the presence and severity of vasculogenic ED and clinical characteristics, and laboratory parameters. We also assessed the association of serum prolidase activity with the variables according to the vascular status of patients with vasculogenic ED. The comparison included 92 vasculogenic ED patients grouped into three categories according to the vascular status of patients with ED; arterial insufficiency (n=26), veno-oclusive dysfunction (n=37), mixed type impotence (n=29) besides 50 controls. Receiver operator characteristics (ROC) were analyzed to find a cut-off value with best sensitivity and lowest false positive rate. Serum prolidase activity (53.5 ± 5.5 vs. 45.7 ± 4.9 U/l, respectively, P <0.001) and serum lipid hydroperoxide levels were significantly increased in patients with vasculogenic ED compared with control cases whereas, serum total free sulfhydryl levels were significantly decreased in patients with vasculogenic ED compared with control cases (P <0.001). The lowest and highest mean serum prolidase activities were detected in control participants and in patients with arterial insufficiency, respectively (ANOVA P<0.001). The overall findings of this study support the predictive accuracy of the serum prolidase activity in our cohort with a statistically significant ROC value of 0.78. Findings of this study have shown that serum prolidase activity is significantly associated with the presence and severity of vasculogenic ED, and elevated serum prolidase activity might be an independent predictor of erectile dysfunction.