Abstract: Background: Behcet’s disease is an autoimmune, recurrent and multisystem disease. Vitamin D has immunomodulator role in immune system. So that vitamin D deficiency was reported in some autoimmune diseases. Behcet’s disease as a Silk Road disease is common in Iran. The aim of this study was to detect the serum level of 25(OH) vitamin D in Behcet’s patients and control group. Methods: In this case–control study, 112 Behcet’s patients as cases group and 112 healthy individuals as controls group were enrolled. Any subject on vitamin D supplement, steroid, and immunosuppressors during the last 6 months were excluded. The serum level of 25(OH) vitamin D was measured in the two groups by ELISA method. The findings were compared via SPSS software. Results: About 57% and 17% of Behcet’s patients had vitamin D deficiency and insufficiency respectively. Vitamin D deficiency was significantly more common in controls than cases group (P < 0.001). Vitamin D levels were significantly lower in controls (P < 0.001). Age and sex did not have any confounding effect on the results. There was no significant relationship between disease duration, disease activity, Pathergy test, HLA-B5, and HLA-B51 with vitamin D level in Behcet’s patients. Conclusions: Vitamin D deficiency is common among Behcet’s patients. However, our results revealed vitamin D deficiency was significantly more common in healthy controls in comparison with Behcet’s cases. Keywords: Behcet’s disease, Internal medicine, Vitamin D