Introduction: Patients with chronic kidney disease (CKD) commonly experience 25-hydroxyvitamin D3 (25-OH-D3) deficiency, and these patients have a higher incidence of cardiovascular diseases (CVDs) due to endothelial dysfunction (ED). The aim of our study was to investigate the effect of 25-OH-D3 deficiency and its supplementation on ED in patients with CKD. Methods: Twenty-nine uremic patients on dialysis and 20 healthy controls were evaluated for ED by high-resolution Doppler ultrasonography of the brachial artery. In addition, 25-OH-D3-deficient patients (25-OH-D3 < 30 nmol/L) with CKD and healthy controls were evaluated for ED before and after 8 weeks of oral vitamin D (cholecalciferol, 50,000 units) treatment. All subjects were evaluated for percent flow-mediated dilatation (% FMD), percent endothelium-independent nitroglycerin-induced vasodilatation (% NID), and bilateral carotid intima-media thickness (CIMT). Findings: Patients on dialysis had lower % FMD and % NID 6.11 [2.27-12.74] and 10.96 [5.43-16.4], respectively, than controls 15.84 [8.19-22.49] and 21.74 [12.49-29.4], respectively (P< 0.05). Patients on dialysis had higher left and right CIMT (0.79 +/- 0.15 and 0.78 +/- 0.14, respectively) than controls (0.60 +/- 0.09 and 0.59 +/- 0.09, respectively; P< 0.05). In 25-OH-D3-deficient patients with CKD, after vitamin D treatment, % FMD was significantly increased in dialysis patients (10.25 [7.8-12.8]) compared to before supplementation (5.4 [2.77-6.15]; P< 0.001). Discussion: These results indicated that dialysis patients had significantly lower blood 25-OH-D3 levels and higher CIMT than healthy subjects. In addition, vitamin D supplementation improved ED and increased % FMD in dialysis patients. Our findings suggest that vitamin D supplementation in dialysis patients might prevent CVD.