Although details of its pathogenesis remain elusive, multiple sclerosis (MS) is now widely accepted as an autoimmune disease of the central nervous system (CNS) in which autoreactive helper T cells play a pivotal role in triggering pathogenic cascades. Recently developed drugs and ongoing clinical trials clearly reflect the significance of targeting pathogenic immune cells, such as T helper 17 (Th17) cells, for MS treatment. Through comprehensive gene expression profiling analysis, we previously showed that the orphan nuclear receptor, NR4A2, is selectively upregulated in peripheral blood T cells from relapsing–remitting MS patients. Furthermore, using experimental autoimmune encephalomyelitis, an animal model of MS, we have shown that NR4A2 is selectively upregulated in peripheral blood T cells and T cells from inflamed CNS tissues. T cells expressing NR4A2 in vivo were induced only when immunized with self-peptide, not with irrelevant exogenous peptides. Accordingly, interleukin-17 (IL-17)-producing helper T cells exclusively express NR4A2, whether or not they secrete interferon (IFN)-γ, suggesting that NR4A2-expressing T cells represent a pathogenic Th17 subset during autoimmunity. Therefore, NR4A2 could be a useful biomarker to estimate pathogenic Th17 cell behavior in MS patients. In addition, a blockade of NR4A2 expression in differentiating Th17 cells with small interfering RNA not only abolished IL-17 secretion, but also Th17-related genes, such as IL-21, c-Maf and IL-23 receptor. Finally, in vivo administration of NR4A2-specific small interfering RNA significantly ameliorated experimental autoimmune encephalomyelitis, implying that NR4A2 is essential for triggering MS/experimental autoimmune encephalomyelitis, and could serve as a novel therapeutic target of the diseases.