Observational studies have suggested that vitamin D may reduce inflammation in relapsing-remitting multiple sclerosis (RRMS), but this has not been clearly confirmed in randomized controlled trials. To further explore the possible anti-inflammatory effects of vitamin D in RRMS, we examined the effect of high-dose oral vitamin D3 on eleven markers of systemic inflammation in 68 RRMS patients enrolled in a double-blinded randomized placebo-controlled trial of vitamin D3supplementation (20,000 IU/week) (NCT00785473). Serum inflammation markers and 25-hydroxyvitamin D (25(OH)D) were measured at baseline and week 96, and no restrictions were set on additional standard immunomodulatory treatment for RRMS. The mean 25(OH)D level rose from 56 ± 29 to 123 ± 34 nmol/L among patients receiving vitamin D3 supplementation, whereas only a minor increase from 57 ± 22 to 63 ± 24 nmol/L was seen in the placebo group. However, no significant differences appeared between the vitamin D group and the placebo group for any of the inflammation markers. Patients on immunomodulatory therapy had significantly higher levels of interleukin-1 receptor antagonist and chemokine (C–X–C motif) ligand 16 than patients without immunomodulatory treatment, but there were no clear synergistic effects between immunomodulatory therapy and vitamin D3 supplementation on any of the inflammation markers. The rise in 25(OH)D levels after vitamin D3 supplementation was unaffected by immunomodulatory treatment. We conclude that in this study of RRMS patients, high-dose oral vitamin D3supplementation prominently increased serum 25(OH)D levels without affecting markers of systemic inflammation, while a more anti-inflammatory phenotype was found among patients on immunomodulatory treatment.