<p>Chronic venous disease and varicose veins of the lower extremities represent a widespread pathology, particularly in individuals with obesity. A high prevalence of varicose vein disease has been observed in obese patients in association with lower plasma levels of vitamin D. The present pilot study aimed to investigate the acute effects of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active form of vitamin D, on oxidative stress in varicose veins obtained from both obese and non-obese patients undergoing cryostripping surgery for varicose vein ablation. Varicose venous samples treated or not with 1,25(OH)₂D₃ (100&#xa0;nM, 12-h incubation) were analysed for reactive oxygen species (ROS) generation using the ferrous xylenol orange oxidation (FOX) assay and immunofluorescence technique. Additionally, the gene expression of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS) was assessed via qPCR. We report a significant reduction in circulating 25-hydroxyvitamin D₃ [25(OH)D₃] levels in obese as compared to non-obese patients. Ex vivo incubation of the venous samples with 1,25(OH)₂D₃ resulted in: (i) significant reduction in ROS level, (ii) upregulation of eNOS and nNOS expression, and (iii) downregulation of iNOS expression in both groups of patients. Vitamin D did not exhibit a ROS scavenger effect, and the antioxidant effect is presumably mediated via its receptor whose presence was confirmed in the varicose venous samples. In conclusion, vitamin D exerts protective effects in venous pathology, which may be beneficial in acute administration prior to the surgical intervention. Large clinical trials are required to assess the optimal dosage and time/duration of administration in patients with chronic venous disease with surgical indication.</p>