Objectives. Determination of vitamin D (VD) status in patients with erectile dysfunction (ED) and testing of the hypothesis that ED patients have lower serum 25(OH)D level in comparison with healthy subjects, as well as that the intensity of ED correlates with 25(OH)D levels. The assumption of possible VD role in ED pathophysiology is based upon previous research on the interplay between common risk factors of ED and other ailments that have been linked to vitamin D deficiency (NVD). Study design. 60 male participants were included in this cross-sectional study: 32 ED patients and 28 control subjects without ED. Patients and Methods. Exclusion criteria were postoperative, anatomical and neurogenic ED, prostate cancer, notable lower urinary tract symptoms and non-approval to participate in the study. After a written informed consent was obtained, anamnesis and physical examination followed, as well as completing the International Index of Erectile Function-5 (IIEF-5) questionnaire, for assessing erectile function and to categorize the study subjects according to ED severity. The blood for 25(OH)D and other laboratory parameters of interest (high- sensitivity C-reactive protein, complete and differential blood count, biochemical, hormonal and electrolyte analysis, glycated hemoglobin and lipid profile) was drawn from the cubital vein, with sampling performed during two winter months of the same year to avoid seasonal variations in cutaneous VD production. Results. Median level of 25(OH)D for all participants was 8.36 ng/mL (interquartile range 4.84 - 11.67 ng/mL), two had insufficient (21 - 29 ng/mL), while all other subjects had deficient VD (< 20 ng/mL). There was no difference in VD levels between ED patients and control subjects (P = 0.894), nor was there any difference when comparing ED groups of disparate severity. 25(OH)D levels did not significantly correlate with the results of IIEF-5 questionnaire. However, patients with both diabetes and ED had lower VD levels in comparison with all other study subjects (P = 0.034). Control subjects without ED had higher parathyroid hormone (PTH) levels compared to ED patients (median 46.8 versus 38 pg/mL, P = 0.031), although PTH levels were normal in general. PTH level positively correlated with 63 better erectile function and in multivariate analysis PTH level showed to be an independent risk factor for ED, lowering ED risk by 4 % with every unit increase. Conclusion. In current research no association between VD status and erectile function was found, except for patients with diabetes and ED, who had lower VD levels compared to all other study subjects. Higher PTH levels in men with normal function of the parathyroid gland appear to have a protective role in preserving the erectile function.