<p>Colorectal cancer with chromosomal instability (CIN⁺) phenotype is immunosuppressive and refractory to immune checkpoint blockade (ICB) therapy. Recently, KIF18A is found to be a mitotic vulnerability in chromosomally unstable cancers, but whether targeting KIF18A affects antitumor immunity in CIN⁺ colorectal cancer is unknown. In our study, western blot, cell viability assay, transwell migration and invasion assays, flow cytometry, animal model, immunohistochemistry (IHC) staining, reverse transcription–quantitative PCR (RT-qPCR) and ELISA assay were conducted to evaluate the potential function of KIF18A in CIN⁺ colorectal cancer. We found that KIF18A inhibition by short hairpin RNAs (ShRNAs) or small inhibitor AM-1882 suppressed proliferation, migration, invasion and tumor growth and metastasis of CIN⁺ colorectal cancer cells in vitro and in vivo. Moreover, targeting KIF18A disrupted cell-cycle progression and induced G2/M arrest in CIN⁺ colorectal cancer cells. In addition, KIF18A inhibition promoted immune infiltration and activation in CIN⁺ colorectal tumors. KIF18A inhibition suppressed proliferation of Tregs and increased infiltration and activation of cytotoxic CD8⁺ T cells in CIN⁺ colorectal tumors. Mechanically, KIF18A inhibition stimulated type I IFN signaling and cGAS-STING activation in CIN⁺ colorectal tumors. Finally, targeting KIF18A enhanced PD-1 blockade efficiency in CIN⁺ colorectal tumors through T cells. Our data elucidated a novel role of KIF18A in antitumor immunity of CIN⁺ colorectal cancer.</p>