Objective ·To explore the phenotype of galectin-9⁺ tumor-associated macrophages (galectin-9⁺TAMs) in muscle-invasive bladder cancer (MIBC). To clarify the regulation of galectin-9⁺TAMs in MIBC microenvironment, and elucidate the mechanism of galectin-9⁺TAMs inhibiting the effector function of CD8⁺T cells and its clinical therapeutic significance in MIBC. Methods ·Phenotype of galectin-9⁺TAMs from MIBC peritumor and tumor tissues was detected by flow cytometry. TCGA (The Cancer Genome Atlas) database was used to sort out the cytokines most relevant to LGALS9 and LGALS9 macrophage gene set. Macrophages were stimulated by recombinant human cytokines in vitro, divided into recombinant human macrophage stimulating factor (rhM-CSF) stimulation group, recombinant human interleukin-16 (rhIL-16) stimulation group and recombinant human interferon-γ (rhIFN-γ) stimulation group. Expression level of galectin-9 among the three groups was verified by flow cytometry. Expression level of galectin-9 on macrophages was detected by flow cytometry after adding M-CSF neutralizing antibody. Effector functions of TAMs were detected by flow cytometry after treating MIBC single cell suspension with anti-galectin-9 antibody. Galectin-9⁺TAMs from peritumor and tumor tissues, and human peripheral blood CD8⁺T cells were sorted and co-cultured in vitro. Effector functions of CD8⁺T cells were detected by flow cytometry. Tumor tissues cultured in vitro were treated with anti-galectin-9 antibody and programmed cell death protein 1 (PD-1) antibody alone or in combination. Tumor cell apoptosis and effector function of CD8⁺T cells were detected by flow cytometry. Results ·Galectin-9⁺TAMs exhibited the phenotype with high expression of human leukocyte antigen DR (HLA-DR), CD86, CD206 and programmed cell death-ligand 1 (PD-L1). Increased IL-10 and transforming growth factor-β (TGF-β) and decreased tumor necrosis factor-α (TNF-α) were secreted by itself. M-CSF, IL-16 and IFN-γ showed the significant difference with LGALS9 and LGALS9 macrophage gene set in TCGA database. Galectin-9 on macrophages increased significantly in rhM-CSF stimulated group and its expression decreased by adding neutralizing antibody. Galectin-9 inhibition switched the activation of TAMs from an immunosuppressive phenotype to a more inflammatory state and PD-L1 on its surface significantly decreased. After cultured in vitro, galectin-9⁺TAMs inhibited the effect of CD8⁺T cells in a partly galectin-9-dependent manner. Compared with applying PD-1 inhibitor alone, percentage of tumor cell apoptosis, the proliferation of CD8⁺T cells and its effector molecules were significantly enhanced or increased in both galectin-9 and PD-1 blockade. Conclusion ·Galectin-9⁺TAMs exhibit an immunosuppressive phenotype and function. Tumor-derived M-CSF induced TAMs to express galectin-9. Galectin-9⁺TAMs inhibit the function of CD8⁺T cells to promote the immune escape of MIBC. Galectin-9 and PD-1 blockade can reactivate the function of CD8⁺T cells more effectively and synergistically.