TGF-β1 induces the conversion of CD4+ CD25-T cells into CD4+ CD25+ Foxp3+ regulatory T cells (Tregs). Galectin-9, one of the β-galactoside-binding animal lectins belonging to the galectin family, induces apoptosis of eosinophils, cancer cells and T cells. In this study, we report the effects of galectin-9 on the conversion of CD4+ CD25-T cells into Foxp3-expressing induced Tregs (iTregs). Galectin-9 together with TGF-β1 had synergistic effects on the rate of conversion to iTregs in vitro. TGF-β1 signaling appears to be essential for the effects of galectin-9 on the generation of iTregs, as galectin-9 promotes TGF-β1-induced phosphorylation of Smad2/3, ERK1/2 and formation of the Smad2/3-Smad4 complex. These data suggest a role for galectin-9 as a promoter of the TGF-β1-dependent conversion of CD4+ CD25-T cells into iTregs in vitro. Therefore, in addition to inducing apoptosis, galectin-9 may contribute to the regulation of inflammation via the expansion of peripheral Tregs.