Abstract Purpose: E75, a peptide derived from the Her2/neu protein, is the most clinically advanced vaccine approach against breast cancer. In this study, we aimed to optimize the E75 vaccine using a delivery vector targeting dendritic cells, the B-subunit of Shiga toxin (STxB), and to assess the role of various parameters (Her2/neu expression, combination with trastuzumab) in the efficacy of this cancer vaccine in a relevant preclinical model. Experimental Design: We compared the differential ability of the free E75 peptide or the STxB-E75 vaccine to elicit CD8+ T cells, and the impact of the vaccine on murine HLA-A2 tumors expressing low or high levels of Her2/neu. Results: STxB-E75 synergized with granulocyte macrophage colony-stimulating factors and CpG and proved to be more efficient than the free E75 peptide in the induction of multifunctional and high-avidity E75-specific anti-CD8+ T cells resulting in a potent tumor protection in HLA-A2 transgenic mice. High expression of HER2/neu inhibited the expression of HLA-class I molecules, leading to a poor recognition of human or murine tumors by E75-specific cytotoxic CD8+ T cells. In line with these results, STxB-E75 preferentially inhibited the growth of HLA-A2 tumors expressing low levels of Her2/neu. Coadministration of anti-Her2/neu mAb potentiated this effect. Conclusions: STxB-E75 vaccine is a potent candidate to be tested in patients with low Her2/neu–expressing tumors. It could also be indicated in patients expressing high levels of Her2/neu and low intratumoral T-cell infiltration to boost the recruitment of T cells—a key parameter in the efficacy of anti-Her2/neu mAb therapy. Clin Cancer Res; 22(16); 4133–44. ©2016 AACR.