Background: The mechanisms by which modifiable environmental and lifestyle factors, including Epstein-Barr virus (EBV) exposure, sun exposure/vitamin D, and smoking exert their effects on multiple sclerosis (MS) risk are unclear. Here, we explored the extent to which differential DNA methylation mediated the associations of previously reported environmental/lifestyle risk factors for first clinical demyelination (FCD). Methods: The Ausimmune case-control study was a multicentre study comprising 282 people recruited soon after an FCD referral and 576 matched-controls. Smoking status, glandular fever history, and recent summer and winter sun exposure were queried. Serum samples were analysed for 25-hydroxyvitmain D (25(OH) D) and anti-EBV serology. Whole-blood EWAS measures were measured using Illumina EPIC BeadChip Array. FCD-associated methylation points (CpG, n=2,432) were inputs to weighted genecorrelation network analysis and 10 CpG clusters were identified. Mediation by dimension-reduced CpG cluster scores was assessed using the MedFlex package in R. Results: Of the 10 CpG clusters, eight were significant mediators of environmental/lifestyle risk factors, indirect effects ranging between 19-34% of EBV, 15-40% of sun exposure, 17-49% of 25(OH)D, and 15-30% of smoking, with some factors acting through common CpG clusters. CpG clusters aligned with pathways involved in signal transduction and transcription regulation, and T-cell activation/proliferation. Discussion: These results demonstrate for the first time that roughly one-third of the associations seen for EBV exposure, sun exposure, 25(OH)D, and smoking are explicable by differential methylation of loci involved in immune cell regulation, providing biologically plausible mechanisms by which these factors can affect MS risk, and suggesting potential points of intervention.