We identified three retinoid-related orphan receptor gamma t (RORt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the ROR binding motif. RORt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.