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Background: Multiple sclerosis (MS) is a demyelinating disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged. In this study, we investigated the possible association of vitamin D levels and rs703842 in the CYP27B1 gene with MS. Methodology: We used blood samples of 99 patients (65 female, 35 male) with an magnetic resonance imaging (MRI)-confirmed definitive diagnosis of MS and 99 controls (70 female, 29 male) between the ages of 18-55 years. We measured their vitamin D levels, isolated their DNA, and scanned rs703842 polymorphism in the CYP27B1 gene. Results: Rs703842 polymorphism in the CYP27B1 gene in humans was found as three different genotypes: CC, CT, and TT. Among them, CC genotype was found higher in the patient group and CT genotype was found higher in the control group. The statistical analysis showed that the probability of a C allele having an association with MS to be 1.5189 times of the probability of T allele. Also, the vitamin D levels in the patient group were detected lower than the control group. Conclusion: Low levels of vitamin D and low expression of CYP27B1 were found to have an association with MS.