Colorectal cancer is one of the most common cancers with a high rate of recurrence, of which current treatments, including surgery, radiation therapy and chemotherapy, have many side effects. Immunotherapy is also a potential therapy for colorectal cancer treatment, which in part relies on the activation of T cell mediated immunity towards antigenic peptides presented on the surface of the cancer cells. These peptides are generated from the degradation of cellular proteins that subsequently bind to and are transported to the cell surface in complex with class I human leucocyte antigens (HLA-I). Such complexes are then scrutinised by T killer cells and in some circumstances, cancer specific antigens are recognised by T killer cells, leading to the eradication of the tumour. In this study, primary and metastatic colorectal cancer cells were used to determine the effect of IFN-γ on immunopeptide presentation. The immunopeptides were investigated following purification of the cell surface peptide-HLA complexes, off-line fractionation by high-performance liquid chromatography (HPLC) and identification by high-resolution mass spectrometry. The results showed IFN-γ treatment increased the diversity of immunopeptides in both primary (~8 fold increase) and metastatic colorectal cancer cells (~21 fold increase). Additionally, incorporating with our proteomics studies from primary and metastatic colorectal cancer cells, the results showed that immunopeptidome of both primary and metastatic colorectal cancer cells is drastically diversified following IFN-γ treatment in the absence of corresponding changes in the proteome. This suggests that IFN-γ treatment enhances antigen processing rather than inducing novel antigen expression, leading to the presentation of novel T cell antigens.