Vitamin D (VD) is recognized as a key immunomodulatory hormone, linking environmental exposure to immune homeostasis. Through activation of the vitamin D receptor (VDR), it regulates transcriptional programs across multiple immune cell types, modulating both innate and adaptive responses. VD limits effector T and B cell differentiation, promotes regulatory subsets, and modulates myeloid cell function. It also reinforces blood–brain barrier integrity, reduces immune cell trafficking, and attenuates microglial and astrocytic activation within the central nervous system. Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system, driven by complex interactions between genetic susceptibility and environmental factors. Among these, VD deficiency is one of the strongest modifiable risk factors and is consistently associated with increased disease activity and disability. The D-Lay MS trial, a randomized, double-blind, placebo-controlled study (n=303), demonstrated that high-dose cholecalciferol (100,000 IU every two weeks) significantly reduced clinicoradiological disease activity over 24 months (HR 0.66, 95% CI 0.50–0.87; p=0.004), supporting a causal contribution of VD in disease modulation. Notably, no protective circulating vitamin D levels could be identified in the treated group, suggesting a saturable immunological effect at the dosing used. At the molecular level, single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from D-Lay MS patients provides in vivo evidence of immune reprogramming. While the frequency of individual lymphocyte subsets remained stable,VD induced substantial transcriptional reprogramming across key pathways, including MAPK, Toll-like receptor, interleukin, and notably NF-κB signaling, particularly in naïve and memory B cells. Epstein–Barr virus (EBV) infection has been described as a necessary driver of MS, relies on NF-κB signaling to maintain infected B cells. VD may interfere with this pathway, providing a mechanistic link between environmental exposure and disease biology. Beyond inflammation, high-dose VD therapy showed a trend toward reduced brain atrophy in D-Lay MS, suggesting potential neuroprotective effects. Its actions on CNS-resident cells further support a role in limiting tissue damage and possibly influencing disease progression. Altogether, these findings position VD as a risk modifier but also a biologically active, clinically relevant immunomodulatory hormone in MS, highlighting its broader therapeutic potential in immune-mediated diseases.