This study aimed to determine the effects of a vitamin D deficiency on aortic functional and structural properties in diabetic rats. Diabetic rats were induced with 50 mg/kg streptozotocin and divided into two equal groups: diabetic rats on a normal diet (DR) and diabetic rats on a vitamin D-deficient diet (DRD). Non-diabetic rats that received a normal diet were the controls (CR). At the end of 10 weeks, rats were sacrificed and aortic rings with and without the endothelium were studied in tissue organ baths for isometric force measurements. Histology of aortic tissue was performed to determine the intima-media thickness. Serum levels of 25-hydroxyvitamin D in the DRD group were significantly decreased compared to the CR and DR groups. Acetylcholine-induced endothelium-mediated relaxation was significantly impaired in DR and DRD compared to CR. Endothelium-dependent contraction to calcium ionophore was significantly augmented in DR and DRD aortas compared to CR. The responses to acetylcholine and calcium ionophore were similar in DRD and DR. There were no significant differences in relaxation to sodium nitroprusside or contraction to phenylephrine between aortas of the groups. The intima-media thickness was significantly greater in the DR group compared to the CR group, and this structural change was augmented in aortas of the DRD group. In conclusion, this study showed that endothelial function was impaired with diabetes, and a vitamin D deficiency did not aggravate endothelial dysfunction. However, diabetes with a vitamin D deficiency demonstrated smooth muscle hypertrophy and an increased aortic media thickness.