© 2021, Fondazione Società Italiana di Neurologia.Background: Patients with multiple sclerosis (MS) have significantly lower vitamin D levels. Cholesterol is known to be the precursor for vitamin D synthesis, and cholesterol removal is regulated by cholesterol 7α-hydroxylase (CYP7A1) in the liver and cholesterol 24S-hydroxylase (CYP46A1) in the brain. In this study, single nucleotide polymorphisms (SNPs) within the genes CYP7A1 (rs3808607) and CYP46A1 (rs754203) were investigated for their effects on serum lipid profiles, vitamin D levels, and the risk of developing MS. Methods: Patients with MS (n = 191) and controls (n = 100) were tested using the PCR–RFLP method to determine their genotypes for rs3808607 and rs754203 SNPs. Results: The minor (C) allele frequency for CYP7A1 rs3808607 variation was 0.380 in patients with MS and 0.305 in control subjects (P =.074). For CYP46A1 rs754203, the frequencies of the minor (C) allele were 0.272 and 0.250 in patients and control subjects, respectively (P =.563). Serum vitamin D (25(OH)D3) concentrations were significantly lower in patients than in control subjects (P =.002). The CYP46A1 rs754203 SNP was associated with total cholesterol levels in patients, whereas the CYP7A1 rs3808607 variant was not associated with serum lipid parameters or vitamin D levels in patients or control subjects. Conclusion: CYP7A1 rs3808607 and CYP46A1 rs754203 variations are not likely to confer an independent risk for MS development in the Turkish population. To the best of our knowledge, this is the first study to investigate the association between CYP46A1 rs754203 and MS risk.