Interleukin 17 (IL-17)-producing T helper cells (T_H-17 cells) are increasingly recognized as key participants in various autoimmune diseases, including multiple sclerosis. Although sets of transcription factors and cytokines are known to regulate T_H-17 differentiation, the role of noncoding RNA is poorly understood. Here we identify a T_H-17 cell–associated microRNA, miR-326, whose expression was highly correlated with disease severity in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis (EAE). In vivo silencing of miR-326 resulted in fewer T_H-17 cells and mild EAE, and its overexpression led to more T_H-17 cells and severe EAE. We also found that miR-326 promoted T_H-17 differentiation by targeting Ets-1, a negative regulator of T_H-17 differentiation. Our data show a critical role for microRNA in T_H-17 differentiation and the pathogenesis of multiple sclerosis.