T helper 17 (Th17) cells that express interleukin-17 (IL-17) play a key role in various inflammatory diseases, such as multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE). The retinoic acid receptor-related orphan receptors γt (RORγt) have an indispensable effect on the differentiation of this cell type, and are thus considered a valuable target in the treatment of Th17-related disorders. In this study, we found that eriodictyol (EDT), a natural flavonoid abundant in citrus fruits and peanuts, was located directly in the binding pocket of RORγt, and induced a conformational change that resulted in the effective suppression of the receptor's activity, thus offering insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, EDT dose-dependently (5-10 μM) blocked murine Th17 differentiation, and markedly reduced IL-17A secretion in vitro. Furthermore, this compound has been found to have novel properties for directly inhibiting Th1 cell development and promoting Treg cell differentiation at high doses (≥10 μM). EDT administration significantly decreased the clinical severity in the EAE model, with inhibited demyelination and reduced inflammatory responses in the periphery and in the central nervous system (CNS). In the adoptive transfer model, EDT also remarkably suppressed the Th17 cell infiltration and pathogenicity. Collectively, our data demonstrated that EDT, as an agent for the pharmacological inhibition of RORγt, has great potential for immunomodulation, and for use in the treatment of Th17-mediated autoimmune disease.