IFN-β is an approved therapeutic option for the treatment of multiple sclerosis. The molecular mechanisms underlying the effects of IFN-β in multiple sclerosis are not fully understood. Migration of dendritic cells (DCs) from the inflammatory site to draining lymph nodes for Ag presentation and activation of naive T cells and to the CNS for reactivation of encephalitogenic T cells requires CCR7 and matrix metalloproteinase (MMP)-9 expression. This article reports for the first time that IFN-β inhibits CCR7 expression and MMP-9 production in mature DCs and reduces their migratory capacity. The effect of IFN-β is mediated through STAT-1. In vivo treatment with IFN-β results in lower numbers of DCs migrating to the draining lymph node following exposure to FITC and in reduced expression of CCR7 and MMP-9 in splenic CD11c+ DCs following LPS administration. IFN-β and IFN-γ share the same properties in terms of their effects on CCR7, MMP-9, and DC migration, but they have opposite effects on IL-12 production. In addition, IFN-β–treated DCs have a significantly reduced capacity for activating CD4+ T cells and generating IFN-γ–producing Th1 cells. The suppression of mature DC migration through negative regulation of CCR7 and MMP-9 expression represents a novel mechanism for the therapeutic effect of IFN-β.