Background <p>Colorectal cancer (CRC) is a highly immunosuppressive malignancy characterized by limited therapeutic options and a poor prognosis. Within the CRC tumor immune microenvironment (TIME), tumor-associated macrophages (TAMs) represent the predominant immune cell population. This study aimed to characterize the specific macrophage subsets contributing to CRC progression and resistance to immunotherapy.</p> Methods <p>Single-cell RNA sequencing (scRNA-seq) was used to identify the key macrophage marker genes. The clinical significance of death-associated protein kinase 1 (DAPK1), was evaluated in clinical CRC cohorts using immunohistochemistry, immunofluorescence, Kaplan-Meier plots and Cox Proportional Hazards Models. Cytometry by time-of-flight, RNA-seq, flow cytometry and an in vitro co-culture system were performed to explore the functional role of macrophage-derived DAPK1 in the TIME. Subcutaneous tumor models and splenic-liver metastasis models were constructed to assess the potential of DAPK1 inhibition to enhance immune checkpoint blockade (ICB) efficacy.</p> Results <p>scRNA-seq analysis revealed predominant DAPK1 expression in intratumoral macrophages of CRC TIME. Elevated macrophage-specific DAPK1 level was significantly associated with worse prognosis. DAPK1⁺TAMs exhibited immunosuppressive properties and inhibit CD8⁺T cells cytotoxicity. Pharmacological inhibition of DAPK1 enhanced the efficacy of anti-PD-1 therapy in murine models.</p> Conclusions <p>DAPK1⁺TAMs infiltration was an independent risk factor for overall survival in patients with CRC. These DAPK1⁺TAMs exhibit an immunosuppressive phenotype and promote the establishment of the TIME with immune evasion. Targeting DAPK1 may represent a potential strategy for synergizing immunotherapy in CRC treatment.</p>