TIM-3, a potential immunotherapeutic target for cancer, has been shown to display diverse characteristics in a context-dependent manner. Thus, it would be useful to delineate the precise functional features of Tim-3 in a given situation. Here, we report that glial TIM-3 shows distinctive properties in the brain tumor microenvironment. TIM-3 was expressed on both growing tumor cells and their surrounding cells including glia and T cells in an orthotopic mouse glioma model. The expression pattern of TIM-3 was distinct from those of other immune checkpoint molecules in tumor-exposed and tumor-infiltrating glia. Comparison of cells from tumor-bearing and contralateral hemispheres of a glioma model showed that TIM-3 expression was lower in tumor-infiltrating CD11b+CD45mid glial cells but higher in tumor-infiltrating CD8+ T cells. In TIM-3 mutant mice with intracellular signaling defects and Cre-inducible TIM-3 mice, TIM-3 affected the expression of several immune-associated molecules including iNOS and PD-L1 in primary glia-exposed conditioned media (CM) from brain tumors. Further, TIM-3 was cross-regulated by TLR2, but not by TLR4, in brain tumor CM- or Pam3CSK4-exposed glia. In addition, following exposure to tumor CM, IFN-gamma production was lower in T cells co-cultured with TIM-3-defective glia than with normal glia. Collectively, these findings suggest that glial TIM-3 actively and distinctively responds to brain tumor, and plays specific intracellular and intercellular immunoregulatory roles that might be different from TIM-3 on T cells in the brain tumor microenvironment.