A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-gamma-producing Tim-3(+) CD8(+) T cells with enhanced granzyme B and perforin expression, although it induced CD4(+) T cell apoptosis. It simultaneously increased the number of Tim-3(+)CD86(+) mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8(+) T cells with DCs from Gal-9-treated mice increased the number of IFN-gamma producing cells and IFN-gamma production. Depletion of Tim-3(+) DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-gamma-producing CD8(+) T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8(+) T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8(+) T cells.