Cholangiocarcinoma (CCA) is a malignant tumor with aggressive biological behavior. Immune checkpoints such as cytotoxic T-lymphocyte antigen 4 (CTLA4) and antiprogrammed death 1 (PD-1) are critical immune-checkpoint molecules that repress T-cell activation. The DNA vaccine potential against CTLA4 and PD-1 in CCA is unknown. We used a thioacetamide (TAA)-induced intrahepatic cholangiocarcinoma (iCCA) rat model to investigate the DNA vaccine potential against CTLA4, PD-1, and PD-L1. We detected PD-L1 expression in CCA and CD8+ T-cell infiltration during CCA progression in rats. We validated antibody production, carcinogenesis, and CD8+ T-cell infiltration in rats receiving DNA vaccination against PD-1, PD-L1, or CTLA4. In our TAA-induced iCCA rat model, the expression of PD-L1 and the infiltration of CD8+ T cells increased as in rat CCA tumorigenesis. PD-1 antibodies in rats were not increased after receiving PD-1 DNA vaccination, and CCA tumor growth was not suppressed. However, in rats receiving PD-L1–CTLA4 DNA vaccination, CCA tumor growth was inhibited, and the antibodies of PD-L1 and CTLA4 were produced. Furthermore, the number of CD8+ T cells was enhanced after PD-L1–CTLA4 DNA vaccination. DNA vaccination targeting CTLA4–PD-L1 triggered the production of specific antibodies and suppressed tumor growth in TAA-induced iCCA rats.