Induced pluripotent stem cells (iPSCs) share transcriptomic similarities with cancer cells and express tumor-specific and tumor-associated antigens, highlighting their potential as cancer vaccines. Our previous study demonstrated that an iPSC-based vaccine effectively prevented tumor growth in various mouse models, including melanoma, breast, lung, and pancreatic cancers. However, the underlying mechanisms and the therapeutic efficacy of the iPSC-based vaccine remain unclear. Colorectal cancer (CRC), the third most common cancer with a rising incidence worldwide, presents an urgent need for novel strategies to prevent and treat CRC. Allograft mouse models were established to evaluate the antitumor effects of the iPSC-based vaccine. CpG oligonucleotide (ODN) 1826 served as a vaccine adjuvant. Bulk RNA-Sequencing (RNA-Seq) and the Microenvironment Cell Population counter (MCP-Counter) algorithm were performed to analyze transcriptomic changes. Liquid chromatography-mass spectrometry (LC-MS) combined with in silico strategies was employed to identify potential antigen proteins. Chinese Hamster Ovary (CHO-K1) models were utilized to express candidate neoantigen proteins. Mouse bone marrow-derived dendritic cells (BMDCs) were used to investigate T cell priming in response to iPSC-associated proteins. Immune cell profiles were characterized by flow cytometry. The combination of CpG and iPSC vaccination demonstrated both prophylactic and therapeutic efficacy in reducing tumor growth in CRC mouse models. Vaccination significantly increased CD8 T cell infiltration within tumor regions, while T cell depletion abrogated the antitumor effects, underscoring the critical role of T cells in mediating these responses. Proteomic analysis identified two iPSC-associated proteins, heterogeneous nuclear ribonucleoprotein U (HNRNPU) and nucleolin (NCL), as key drivers of the observed immune responses. Vaccination with HNRNPU or NCL, in combination with CpG, enhanced dendritic cell activation, induced antigen-specific CD8 T cell cytotoxicity, and promoted the formation of central memory CD8 T cells, collectively leading to significant CRC tumor shrinkage. Our findings reveal potential mechanisms underlying the efficacy of iPSC-based vaccines in cancer immunotherapy. Additionally, HNRNPU and NCL were identified as key antigen proteins in iPSC, demonstrating promise for the development of peptide-based vaccines for both the prevention and treatment of CRC.