The Tim3/Gal9 pathway is associated with immunosuppression and worse clinical outcome in multiple cancers. To illustrate the specific mechanism of Tim3/Gal9 interaction in osteosarcoma, we examined expression, function, and regulation of Tim3/Gal9 in various cells from osteosarcoma patients. Data showed that CD4+ T cells, CD8+ T cells, and monocytes from both peripheral blood and tumor of osteosarcoma patients contained high frequencies of Tim3+ cells, while the Gal9 expression was primarily found in regulatory T cells (Tregs) from osteosarcoma patients and was elevated compared to that in non-cancer controls. The Tim3+ CD4+ and CD8+ T cells presented lower proliferation capacity compared to their Tim3− counterparts, which could be reverted by blocking Tim3 or Gal9. Interestingly, purified Tim3+ CD4+ T cells secreted more interferon gamma (IFNγ) than purified Tim3− CD4+ T cells, but IFNγ production by Tim3+ CD4+ T cells was vulnerable to Gal9-mediated suppression. In monocytes, Tim3 expression was associated with high interleukin (IL)-10 and low IL-12 cytokine secretion profile. Exogenous recombinant Gal9, as well as CD4+CD25+ Treg supernatant, further decreased IL-12 expression in monocytes. In CD4+ T cell-monocyte coculture experiments, Tim3+ monocytes inhibited IFNγ expression from total CD4+ T cells and the development of IFNγ response in naive CD4+ T cells. Blocking the Tim3/Gal9 pathway reverted these effects. Together, these results suggested that in osteosarcoma patients, Tim3 expression did not directly mediate immune suppression, but the interaction between Tim3+ T cells and monocytes, naive CD4+ T cells, and Gal9-expressing CD4+CD25+ Tregs could resulting in progressive suppression of Th1 responses.