Purpose: HER2/neu, a source of immunogenic peptides, is expressed in >75% of breast cancer patients. We have conducted clinical trials with the HER2/neu E75 peptide vaccine in breast cancer patients with varying levels of HER2/neu expression. Vaccine response based on HER2/neu expression level was analyzed. Experimental Design: Patients were stratified by HER2/neu expression. Low expressors (n = 100) were defined as HER2/neu immunohistochemistry (IHC) 1 + to 2 + or fluorescence in situ hybridization < 2.0. Overexpressors (n = 51) were defined as IHC 3 + or fluorescence in situ hybridization ≥ 2.0. Additional analyses were done stratifying by IHC status (0-3 +). Standard clinocopathlogic factors, immunologic response (in vivo delayed-type hypersensitivity reactions; ex vivo human leukocyte antigen A2:immunoglobulin G dimer assay), and clinical responses (recurrence; mortality) were assessed. Results: Low-expressor (control, 44; vaccinated, 56) versus overexpressor patients (control, 22; vaccinated, 29) were assessed. Low expressors, overexpressors, and most IHC-status vaccinated groups responded immunologically.Vaccinated low-expressor patients had larger maximum immunologic responses compared with overexpressor patients (P = 0.04), andvaccinated IHC1 + patients had increased long-term immune response (P = 0.08). More importantly, compared with controls, low-expressor patients had a mortality reduction (P = 0.08). The largest decrease in mortality was seen in IHC 1 + patients (P = 0.05). In addition, a subset of overexpressor patients (n = 7) received trastuzumab before vaccination, and this combination seems safe and immunologically beneficial. Conclusions: Most patients with various levels of HER2/neu expression responded immunologically and seemed to benefit from vaccination. The low expressors, specifically IHC1 + patients, had more robust immunologic responses and may derive the greatest clinical benefit from the E75 vaccine.