Type I interferons (IFNs) have shown therapeutic potential in treating CNS autoimmune diseases (IFN-β for multiple sclerosis and IFN-α for uveitis), but treatment is not always effective. Here, we describe a non-redundant requirement for GαI-coupled CXCR3 in the immunomodulatory actions of type I IFNs that culminates in the suppression of human uveitis and experimental autoimmune uveitis (EAU). Effective treatment with IFN-α/β inhibited autopathogenic CD4+ T cell migration to effector sites in mice by upregulating expression of the cognate ligands CXCL9, CXCL10 and CXCL11, causing ligand-mediated downregulation of CXCR3 expression and effector T cell retention in the spleen. These effects of IFN-α/β also required IFN-γ. In the absence of CXCR3 signaling, type I IFN was ineffective in active EAU. In patients with uveitis, disease exacerbations associated with reduced serum IFN-α concentrations. Importantly, IFN-α/β reduced CXCR3 expression and human effector T cell migration, and these parameters markedly associated with IFN-α therapeutic efficacy in uveitis patients. Our findings provide new insights into the molecular basis of type I IFN therapy for CNS autoimmune diseases and identify CXCR3 as a critical biomarker for effective immunotherapy with type I IFNs.