Introduction: The impact of interactions between the two estrogen receptor (ER) subtypes, ER(alpha) and ER(beta), on gene expression in breast cancer biology is not clear. The goal of this study was to examine transcriptomic alterations in cancer cells co-expressing both receptors and the association of gene expression signatures with disease outcome. Methods: Transcriptional effects of ER(beta) overexpression were determined in a stably transfected cell line derived from ER(alpha) -positive T-47D cells. Microarray analysis was carried out to identify differential gene expression in the cell line, and expression of key genes was validated by quantitative polymerase chain reaction. Microarray and clinical data from patient samples were then assessed to determine the in vivo relevance of the expression profiles observed in the cell line. Results A subset of 14 DNA replication and cell cycle-related genes was found to be specifically downregulated by ER(beta). Expression profiles of four genes,CDC2, CDC6, CKS2, and DNA2L, were significantly inversely correlated with ER(beta) transcript levels in patient samples, consistent with in vitro observations. Kaplan-Meier analysis revealed better disease outcome for the patient group with an expression signature linked to higher ER(beta) expression as compared to the lower ER(beta)-expressing group for both disease-free survival (p = 0.00165) and disease-specific survival (p = 0.0268). These findings were further validated in an independent cohort. Conclusion: Our findings revealed a transcriptionally regulated mechanism for the previously described growth inhibitory effects of ER(beta) in ER(alpha)-positive breast tumor cells and provide evidence for a functional and beneficial impact of ER(beta) in primary breast tumors.