The interaction between T cell immunoglobulin‐ and mucin‐domain‐containing molecule (Tim‐3) expressed on T helper 1 (Th1) cells, and its ligand, galectin‐9, negatively regulates Th1‐mediated immune responses. However, it is poorly understood if and how the Tim‐3/galectin‐9 signaling pathway is involved in immune escape in patients with hepatocellular carcinoma (HCC). Here we studied the expression, function, and regulation of the Tim‐3/galectin‐9 pathway in patients with hepatitis B virus (HBV)‐associated HCC. We detected different levels of galectin‐9 expression on antigen‐presenting cell (APC) subsets including Kupffer cells (KCs), myeloid dendritic cells (DCs), and plasmacytoid DCs in HCC. The highest galectin‐9 expression was on KCs in HCC islets, not in the adjacent tissues. Furthermore, Tim‐3 expression was increased on CD4⁺ and CD8⁺ T cells in HCC as compared to the adjacent tissues, and Tim‐3⁺ T cells were replicative senescent and expressed surface and genetic markers for senescence. Interestingly, tumor‐infiltrating T‐cell‐derived interferon (IFN)‐γ stimulated the expression of galectin‐9 on APCs in the HCC microenvironment. Immunofluorescence staining revealed a colocalization of Tim‐3⁺ T cells and galectin‐9⁺ KCs in HCC. Functional studies demonstrated that blockade of the Tim‐3/galectin‐9 signaling pathway importantly increased the functionality of tumor‐infiltrating Tim‐3⁺ T cells as shown by increased T‐cell proliferation and effector cytokine production. Finally, we show that the numbers of Tim‐3⁺ tumor‐infiltrating cells were negatively associated with patient survival. Conclusion: Our work demonstrates that the Tim‐3/galectin‐9 signaling pathway mediates T‐cell senescence in HBV‐associated HCC. The data suggest that this pathway could be an immunotherapeutic target in patients with HBV‐associated HCC. (HEPATOLOGY 2012)