It remains a critical issue to improve the survival rate in patients with recurrent or metastatic breast cancer. This study sought to develop a prognostic scheme based on a 28-gene signature in a broad patient population, including those with advanced disease. Clinically annotated transcriptional profiles of 1,734 breast cancer patients were obtained to validate the 28-gene signature in prognostic categorization. The 28-gene signature generated significant patient stratification with regard to breast cancer disease-free survival (log-rank P<0.0001; n=1,337) and overall survival (log-rank P<0.0001; n=806) in Kaplan-Meier analyses. The gene expression signature provides refined prognosis of disease-free survival (log-rank P<0.006; Kaplan-Meier analysis) within each classic clinicopathologic factor-defined subgroup, including LN-, LN+, ER-, ER+ and tumor grade II. Furthermore, it was investigated whether this gene signature predicts chemoresponse to drugs commonly used to treat breast cancer. The mRNA expression levels of this gene signature in NCI-60 cell lines were used to predict chemoresponse to CMF, tamoxifen, paclitaxel, docetaxel, and doxorubicin (adriamycin). The 28-gene prognostic signature accurately (P<0.02) predicted chemotherapeutic response to the studied drugs. This study confirmed the prognostic applicability of the breast cancer gene signature in a broad clinical setting. This prognostic signature is also predictive of drug response in cancer cell lines.