Background: There is a known inverse association between solar radiation and the prevalence of multiple sclerosis (MS). Some studies have investigated the link between vitamin D and MS. The aim of this study was to investigate the possible association between serum 25(OH) vitamin D3 concentration and the severity of disease in Iranian patients with MS.Methods:Patients with relapsing-remitting MS underwent neurological examination, including measurement of Expanded Disability Status Scale (EDSS) score, and were categorized by disease severity into mild (0 ≤ EDSS ≤3), moderate (3.5 ≤ EDSS ≤5.5) and severe (6 ≤ EDSS). Serum concentrations of 25(OH) vitamin D3, calcium, phosphorus, magnesium and parathyroid hormone were also measured.Results:A total of 78 (73.1% female) patients with MS were evaluated. The mean (± standard deviation) of age was 33.9 ± 9.2 years. The mean (± standard error) serum concentrations of 25(OH) vitamin D3 were 36.6 ± 5.1 mg/dL, 50.1 ± 12.6 mg/dL and 19.8 ± 6.5 mg/dL in patients with mild, moderate and severe disease, respectively. There was a statistically significant inverse correlation between 25(OH) vitamin D3 concentration and EDSS score (P = 0.016, r= -0.273 by Spearman rank correlation test), which was observed in women only (P = 0.021, r = -0.305). Receiver operating characteristic curve analysis suggested that a serum 25(OH) vitamin D3 concentration cutoff of 16.5 mg/dL could differentiate patients with mild/moderate MS from severe disease with 74.6% accuracy.Conclusion:Our findings further support the association between vitamin D and disease severity in MS.