Breast cancer is the leading cause of cancer-associated deaths in women. Combination immunotherapy attracts great interest as a treatment for breast cancer. However, there are no studies on the use of cytotoxic T-lymphocyte antigen 4 (CTLA-4) monoclonal antibody in combination with the melanoma-associated antigen A family (MAGE-As) co-antigen peptide (p248V9) for treating breast cancer, which should be explored. To this aim, in the present study, the samples of 115 patients with breast cancer were collected, and MAGE-As and CTLA-4 levels in breast cancer and adjacent normal tissues were assessed by immunohistochemical staining. The effect of 5-aza-2 '-deoxycytidine (5DC) on the expression of MAGE-As in breast cancer cell lines was assessed by reverse transcription-quantitative PCR and western blot assay. Cytotoxic T cells (CTLs) were induced by MAGE-As co-antigen peptide. The specific lytic rate and IFN-gamma level were examined by CCK-8 assay and ELISA, respectively. It was found that MAGE-As were highly expressed in breast cancer tissues. 5DC treatment promoted the expression of MAGE-As in breast cancer cells. The upregulation of the expression of MAGE-As specifically enhanced the ability of CTLs to kill breast cancer cells. CTLA-4 was highly expressed in breast cancer tissues and cells, and patients with breast cancer exhibiting high expression of CTLA-4 had low overall survival. CTLA-4 promoted the lytic efficiency of CTLs in breast cancer cells, and the combination of an anti-CTLA-4 antibody and 10 mu M 5DC exhibited the highest cell lysis ability of CTLs. The present study demonstrated that MAGE-As co-antigen peptide-specific CTLs in combination with an anti-CTLA-4 monoclonal antibody and 5DC, have potent tumor cell-killing effects. It provides a novel theory for the development of breast cancer therapies.