In autoimmune hepatitis (AIH), liver-damaging CD4 T cell responses are associated with defective CD4posCD25pos regulatory T cells (T-regs). Galectin-9 (Gal9), a β-galactosidase–binding protein expressed by T-regs, is key to their function, inhibiting T helper 1 immune responses by binding T cell immunoglobulin and mucin domain 3 (Tim-3) on CD4 effector cells. We investigated whether impaired immunoregulation in AIH results from reduced expression of Gal9 in T-regs and/or Tim-3 on CD4 effector cells. Circulating Gal9posCD4posCD25pos and Tim-3posCD4posCD25neg T cell phenotype was assessed by flow cytometry in 75 AIH patients. To evaluate whether Tim-3 expression renders CD4posCD25neg T cells amenable to T-reg control, purified CD4posCD25negTim-3pos (Tim-3pos) and CD4posCD25negTim-3neg (Tim-3neg) cells were cocultured with T-regs. To determine whether Gal9 expression is essential to function, T-regs were treated with small interfering RNA (siRNA) to repress Gal-9 translation; T-reg suppressor function was assessed by proliferation. In AIH, Tim-3pos cells within CD4posCD25neg cells and their T-betpos and RORCpos subsets were fewer and contained higher numbers of interferon-γ (IFNγ)pos and interleukin (IL)-17pos cells than healthy subjects (HS). In AIH and HS, Tim-3pos cells proliferated less vigorously and were more susceptible to T-reg control than Tim-3neg cells. In AIH, Gal9posT-regs were fewer and contained less FOXP3pos, IL-10pos, and transforming growth factor βpos and more IFNγpos and IL-17pos cells than HS. siRNA treatment of Gal-9pos T-regs drastically reduced T-reg ability to suppress CD4posCD25neg and Tim-3pos cell proliferation in AIH and HS. Tim-3pos cell percentage correlated inversely with aminotransferase and CD25negT-betpos cell values. Conclusion: Reduced levels of Tim-3 on CD4posCD25neg effector cells and of Gal9 in T-regs contribute to impaired immunoregulation in AIH by rendering effector cells less prone to T-reg control and T-regs less capable of suppressing. (HEPATOLOGY 2012)