Background and purpose High levels of 25-hydroxyvitamin D-3 (25[OH]D-3) are associated with a lower risk for multiple sclerosis (MS). The bioavailability of 25(OH)D-3 is regulated by its main plasma carrier, vitamin D-binding protein (DBP). Free 25(OH)D-3 can be estimated by also measuring DBP concentration. In addition, DBP has immunomodulatory functions that may independently affect MS pathogenesis. No previous studies have assessed free 25(OH)D-3 or DBP in presymptomatically collected samples. This study was undertaken to assess free 25(OH)D-3 and DBP as risk factors for MS. Methods A nested case-control study was performed with presymptomatic serum samples identified through cross-linkage of MS registries and Swedish biobanks. Concentration of 25(OH)D-3 was measured with liquid chromatography and DBP levels with sandwich immunoassay. Free 25(OH)D-3 was approximated as free vitamin D-3 index: (25[OH]D-3/DBP) x 10(3). MS risk was analyzed by conditional logistic regression, calculating odds ratios (ORs) with 95% confidence intervals (CIs). Results Serum samples from 660 pairs of matched cases and controls were included. At <20 years of age, high levels of free vitamin D-3 index were associated with a lower risk of MS (highest vs. lowest quintile: OR = 0.37, 95% CI = 0.15-0.91, p for trend across quintiles = 0.04). At age 30-39 years, high levels of DBP were associated with a lower MS risk (highest vs. lowest quintile: OR = 0.36, 95% CI = 0.15-0.85, p for trend = 0.02). Conclusions These findings support the hypothesis that high levels of free 25(OH)D-3 at a young age reduce the risk of MS later in life. They also implicate a role for DBP in MS etiology.