Interferon (IFN)beta has been used over the past decades as an effective first-line therapy against relapsing-remitting multiple sclerosis (RR MS), however its in vivo operative mechanisms of action are not fully understood. Current advances in our understanding of the development of the autoimmune response, including its induction by a recently discovered Th17 cell lineage, may allow us to identify the biomarkers of this effective therapy. Our in vitro human studies have characterized IFNbeta's immunoregulatory effects on Th17 cell differentiation. IFNbeta inhibited IL-1beta, IL-23 and transforming growth factor (TGF)-beta (which induce Th17 cell differentiation), and induced IL-27, IL-12p35 and IL-10 (which suppress it) in dendritic cells (DCs) and B-cells. The effect on IL-1beta, IL-23 and IL-27 production in DCs was mediated by the up-regulation of Toll-like receptor (TLR)7 and its downstream signaling molecules. IFNbeta's direct effect on naïve T-cells suppressed in vitro Th17 differentiation by inhibiting Th17 cell lineage markers (retinoic acid-related orphan nuclear hormone receptor (ROR)c, IL-17A, IL-23R and CCR6), and by inducing IL-10 production by CD4 cells, which constrains Th17 cell expansion. Our results have identified novel therapeutic mechanisms of IFNbeta, which inhibit Th17 cell differentiation in the context of the autoimmune response in MS.