Th17 cells, a lymphocyte subpopulation that is characterized by the expression of the transcription factor "retinoic acid receptor-related orphan receptor gamma-t" (RORγt), plays an important role in the pathogenesis of autoimmune disease. The current study was set up to discover novel and non-steroidal small-molecule inverse agonists of RORγt and to determine their effects on autoimmune disease. Structure-based virtual screening (SBVS) was used to find compounds targeting RORγt. Flow cytometry was used to detect the Th17 cell differentiation. Inverse agonists were intraperitoneally administered to mice undergoing experimental autoimmune uveitis (EAU), experimental autoimmune encephalomyelitis (EAE) or type 1 diabetes. The effects of the inverse agonists were evaluated by clinical or histopathological scoring. Among 1.3 million compounds screened, CQMU151 and CQMU152 were found to inhibit Th17 cell differentiation without affecting the differentiation of Th1 and Treg lineages (both P = 0.001). These compounds also reduced the severity of EAU (P = 0.01 and 0.013) and functional studies showed that they reduced the number of Th17 cell and the expression of IL-17(Th17), but not IFN-γ(Th1) and TGF-β(Treg) in mouse retinas. Further studies showed that these compounds may reduce the expression of p-STAT3 by reducing the positive feedback loop of IL-17/IL-6/STAT3. These compounds also reduced the impaired blood-retinal barrier function by upregulating the expression of tight junction proteins. These compounds were also found to reduce the severity of EAE and type 1 diabetes. Our results showed that RORγt inverse agonists may inhibit the development of autoimmune diseases and may provide new clues for the treatment of Th17-mediated immune diseases.