The purpose of this study was to explore the ameliorative effect of CpG-ODN on spleen damage after carbon ion radiation (CIR). In the experiment, C57BL/6L mice were irradiated with 5 Gy of CIR and the index of spleen was calculated. Changes in spleen tissue structure were observed by histological examination (HE). Double-strand break (DSB) of DNA and apoptosis were detected by γ-H₂AX immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay, respectively. The number of B cells in the peripheral blood was examined using CD19 antibody labeling. These results showed that CpG-ODN improved the index of spleen, relieved red pulp atrophy, and increased the area of white pulp, in which the number of γ-H₂AX foci and TUNEL-positive cells were decreased after CIR. In addition, CpG-OND increased the number of CD19⁺ B cells in peripheral blood after CIR. These results indicated that CpG-ODN ameliorates spleen damage induced by CIR, possibly by inhibiting the rate of DSB and apoptosis.