CD4⁺ T helper lymphocytes that express interleukin-17 (T_H17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T_H17 cell differentiation and function with susceptibility to Crohn’s disease, rheumatoid arthritis and psoriasis^,,. Thus, the pathway towards differentiation of T_H17 cells and, perhaps, of related innate lymphoid cells with similar effector functions^,, is an attractive target for therapeutic applications. Mouse and human T_H17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T_H17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T_H17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4⁺ T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.