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Background Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) is a synthetic single-stranded DNA and immunomodulator. In the murine tumor, CpG ODN suppresses tumor growth and increases CD8 + T cell infiltration. This study aimed to elucidate the immune landscape that underly tumor suppression associated with CpG ODN at the single-cell level.Methods We orthotopically injected syngeneic tumor cell line BNL into BALB/c mice and intravenously injected CpG ODN or a control ODN at days 7 and 14. We collected CD45 + immune cells at days 21 and sent for CITE-Seq single-cell sequencing. We focused on 14,209 cells passed quality control filters. Data from RNA and antibody-derived tags was integrated with weighted nearest neighbor to perform unsupervised clustering and visualization.Results We identified 5 major clusters, namely B cell, T/NK cell, monocyte/macrophage, neutrophil, and basophil. Significant increases in activated neutrophils and cytotoxic T cell subclusters were found in the CpG-ODN-treated liver tumors. Differential gene expression analysis suggested a distinct transcriptomic progression and pathway activation of neutrophil following CpG ODN treatment. We also observed significant clonal expansion of cytotoxic T cell subclusters in the CpG-ODN-treated liver tumors. Cell-cell interaction analysis disclosed frequent interaction between cytotoxic T cells and activated neutrophils.Conclusions We demonstrated that CpG ODN treatment can induce activation of neutrophils and expansion of cytotoxic T cells. Our study provides insight into how CpG ODN treatment augment T cell cytotoxicity to achieve tumor suppression.