The loss of PTEN and phosphorylated Akt (pAkt) expression is thought to be involved in the mechanism leading to trastuzumab resistance in patients with HER2-positive breast cancer. We retrospectively performed immunohistochemical analyses for estrogen receptor, progesterone receptor, HER2/neu, PTEN, pAkt, and p53 expression in tumor specimens obtained before and after trastuzumab-containing neo-adjuvant chemotherapy. The intensity of staining was evaluated for each biomarker, and the correlations between the immunohistochemical profiles and the clinical outcome were analyzed. The changes in the immunohistochemical profiles between specimens obtained before and after trastuzumab-containing neo-adjuvant chemotherapy were evaluated for patients with residual tumors. The present study included 44 patients with breast cancer who received trastuzumab-containing neo-adjuvant chemotherapy. Seventeen patients achieved a pathological complete response. The patients were positive for PTEN and pAkt (PTEN = 14%, N = 6/44; pAkt, 80%, N = 35/44). The expression of both PTEN and pAkt were not correlated with pathological complete response. Persistent HER2/neu over-expression after neo-adjuvant chemotherapy was significantly associated with recurrence. Among 27 patients with residual cancer, the percentages of patients with HER2/neu-positive or pAkt-positive tumors were low, but PTEN expression was elevated. The present study suggested that neither the immunohistochemical expression of PTEN nor the expression of pAkt was associated with the clinical outcome of trastuzumab-containing neo-adjuvant chemotherapy. Except among patients with pathological complete remission, the persistent over-expression of HER2/neu may be a poor prognostic factor.