Although peptide vaccines offer a novel venue for cancer immunotherapy, clinical success has been rather limited. Cell-penetrating peptides, due to their ability to translocate through the cell membrane, could be conjugated to the peptide vaccine to2 enhance therapeutic efficiency. The S4 transduction domain of the shaker-potassium channel was conjugated to mammaglobin-A (MamA) immunodominant epitope (MamA2.1) to verify its anticancer immunogenicity. S4-MamA2.1 peptide has demonstrated significantly higher epitope loading and stable membrane expression of HLA-A2 antigen-presenting molecules on T2 cell lines. Further, these S4-MamA2.1 treated T2 cells were able to activate naïve CD8+ T cells to induce MamA-specific cytotoxicity against breast cancer cells. Conjugation of the S4 domain has also demonstrated a slight increase in immunogenicity of lesser immunodominant MamA epitopes. The conjugation of the S4 domain to N-terminus of MamA2.1 demonstrated significantly higher immunogenicity over C-terminus conjugation. Taken together, the results of the present study suggest that conjugation of the S4 cell-penetrating peptide domain to MamA2.1 epitope enhances the peptide vaccine immunogenicity against MamA-expressing breast cancers.