Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4⁺ T cells and the structure of its T cell epitope was determined by proteomics-based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I-A^k/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH-2 mouse HCC cells. MHC class II-binding peptides found in MIH-2-loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276–290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)-γ production of splenocytes and suppressed the growth of implanted CYP2J-positive MIH-2 cells but not CYP2J-negative murine bladder tumour cells. However, continuous treatment of MIH-2-bearing mice with CYP2J peptide significantly suppressed IFN-γ production of splenocytes and accelerated the growth of implanted MIH-2 tumours in vivo. Increased frequencies of CD4⁺forkhead box P3 regulatory T cells and CD11b⁺Gr-1⁺ myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti-tumour immunity at an initial stage of HCC, but suppress host anti-tumour immunity with excessive antigenic stimulation at an advanced stage.