Background Vitamin D deficiency is a major health concern as it increases the risk of developing many serious diseases. Recently, the correlation between vitamin D deficiency and multiple sclerosis (MS) is a matter of serious debate. In this case-control study, we aimed to assess the correlation between genetic changes in the vitamin D-binding protein (DBP) gene and their consequence on MS patients. Our sample study consisted of 110 individuals; 40 patients with MS as cases and 70 healthy controls. Vitamin D levels were determined by immunofluorescence assay, and polymorphisms at rs7041 (c.1296 T > G p.Asp416Glu) and rs4588 (c.1307C > A p.Thr420Lys) of the DBP gene were genotyped using PCR/RFLP method for all cases and controls. Results Our results showed that genotype frequencies were consistent with Hardy-Weinberg equilibrium. A significant association was found in rs7041 (c.1296TT) homozygous wild-type, and the odds ratio was < 1 suggesting a protective role against developing MS (OR; 0.03, p = 0.0002) whereas the c.1296GG genotype was significantly correlated with an increased risk for MS by 6 folds (OR: 6.0000, p < 0.0001). No significant association was noted at rs4588 and MS occurrence. In addition, our compound genotyping results revealed that haplotypes 1S-1S are 6 times more likely to develop MS, whereas haplotypes 1F-1F had a more protective role in MS patients (OR: 0.063, p = 0.06.), respectively. The risk of vitamin D insufficiency in patients was greater by 14 folds compared to controls (OR: 14.05, p = 0.0128). Furthermore, the c.1296GG genotype was associated significantly by more than 4 times with insufficient levels of vitamin D and by 7 folds with vitamin deficiency. Conclusions We conclude that polymorphisms in the DBP gene could have independent effects on the risk of developing multiple sclerosis. The homozygous recessive genotype at rs7041 was associated with insufficient levels of vitamin D and with the risk of MS emergence.