Overexpression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is related to the exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) in diffuse large B-cell lymphoma (DLBCL). However, the mechanism of TIM3-mediated CD8+TILs exhaustion in DLBCL remains poorly understood. Therefore, we aimed to clarify the potential pathway involved in TIM3-mediated CD8+TILs exhaustion and its significance in DLBCL.The expression of TIM3 and its correlation with CD8+TILs exhaustion, the key ligand of TIM3, and the potential pathway of TIM3-mediated CD8+TILs exhaustion in DLBCL were analyzed using single-cell RNA sequencing and validated by RNA sequencing. The biological significance of TIM3-related pathway in DLBCL was investigated based on RNA sequencing, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction data. Finally, the possible regulatory mechanism of TIM3-related pathway in DLBCL was explored using single-cell RNA sequencing and RNA sequencing.Our results demonstrated that CD8+TILs, especially the terminally exhausted state, were the major clusters that expressed TIM3 in DLBCL. Galectin-9, mainly expressed in M2 macrophages, is the key ligand of TIM3 and can induce the exhaustion of CD8+TILs through TIM3/Galectin-9 pathway. Meanwhile, high TIM3/Galectin-9 enrichment is related to immunosuppressive tumor microenvironment, severe clinical manifestations, inferior prognosis, and poor response to CHOP-based chemotherapy, and can predict the clinical efficacy of immune checkpoint blockade therapy in DLBCL. Furthermore, the TIM3/Galectin-9 enrichment in DLBCL may be regulated by the IFN-γ signaling pathway.Our study highlights that TIM3/Galectin-9 pathway plays a crucial role in CD8+TILs exhaustion and the immune escape of DLBCL, which facilitates further functional studies and could provide a theoretical basis for the development of novel immunotherapy in DLBCL.© 2024. The Author(s).