Objective <p>Developing effective therapeutic strategies for colorectal cancer (CRC) presents a significant clinical challenge. Although immune checkpoint blockade therapies, such as the CD47 blockade, have shown promise, their efficacy is often limited by suboptimal responses and associated toxicities. This study explores the potential of the gut microbial metabolite cyclic diadenosine monophosphate (c-di-AMP) to enhance the therapeutic efficacy of CD47 antibodies in treating CRC while mitigating its adverse effects.</p> Methods <p>A subcutaneous tumor model was established to assess the therapeutic efficacy of c-di-AMP in combination with CD47 blockade for CRC treatment. Growth inhibition assays were performed to evaluate the direct effects of c-di-AMP on CRC cells. Cell removal assays, flow cytometry, and immunofluorescence were employed to investigate the impact of c-di-AMP on macrophage-mediated phagocytosis. Macrophage polarization phenotypes were characterized by flow cytometry and immunohistochemistry. Transcriptome sequencing was also conducted to elucidate the molecular mechanisms underlying the c-di-AMP-induced enhancement of macrophage phagocytic activity.</p> Results <p>Our results revealed that c-di-AMP, in combination with CD47 blockade, exerted potent anticancer effects in vivo. Mechanistically, c-di-AMP enhanced macrophage-mediated phagocytosis triggered by CD47 blockade by promoting M1 polarization and activating the NF-κB signaling pathway. Furthermore, c-di-AMP upregulated the expression of CD11b/CD18, thereby strengthening intercellular adhesion between macrophages and CRC cells, which further facilitated phagocytosis.</p> Conclusion <p>These findings indicate that c-di-AMP may enhance the efficacy of CD47 blockade in CRC by promoting macrophage phagocytosis through NF-κB activation.</p>