Vitamin D deficiency is associated with cardiovascular and metabolic diseases. Cardiovascular diseases, in turn, are responsible for mortality of patients with type 2 diabetes (T2D). We investigated whether a single parenteral dose of 25(OH) Vit D could improve the endothelial function in T2D patients with ischemic heart disease. A randomized, placebo-controlled, and double-blind trial was performed on 112 patients randomly divided into vitamin D (n = 55) and placebo (n = 57) groups. A randomization table was used to administer a single dose of either vitamin D (300000 IU) or a matching placebo, intramuscularly. The levels of 25(OH) Vit D, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) were measured at baseline and at 8 weeks. In the supplemented group, the level of serum 25(OH) Vit D was increased significantly (29.6 ± 20.8 44.5 ± 19.2 ng/mL; < 0.05), whereas no changes were observed in the placebo group. Within the supplemented group, before and after vitamin D intervention no significant changes in the levels of ICAM-1 and VCAM-1 were observed. The marginal means of the outcome variables (ICAM-1, VCAM-1, and 25(OH) Vit D) were compared between the groups using ANCOVA, adjusted for the baseline of each variable itself: no significant difference was seen in the markers of the endothelial function. A single parenteral dose of vitamin D in T2D patients with ischemic heart disease failed to show improvement in endothelial function.