Estrogen receptor (ER) is regarded as a major causal factor in breast cancer and FoxA1, a winged-helix transcription factor belonging to the forkhead family, has been found to function as a pioneer factor in the recruitment of ER to several cis-regulatory elements in the genome. High throughput chromatin immunoprecipitation analyzed by hybridization to microarrays (ChIP-chip) can reveal ER and FoxA1 binding sites occupied by transcription factors. However, these results are blind to lineage-specific cis-regulatory elements. In this study, we identified ER and FoxA1 binding sites conserved in evolution by using Detection of LinEage-Specific Selection (DLESS) method. We also analyzed target genes close to conserved cis-regulatory elements by combining gene-expression data. A total of 7877 ER binding sites and 18,135 FoxA1 binding sites were identified in MCF-7 cells by performing an unbiased genome-wide ChIP-chip with False Discovery Rate (FDR) of 5%. Using DLESS method, we found target candidate genes closest to fully conserved cis-regulatory elements related to apoptosis according to gene ontology analysis. Furthermore, network analysis of apoptosis-related genes within 10 kb of fully conserved cis-regulatory elements was constructed using Ingenuity Pathway Analysis (IPA). Apoptosis genes in the network showed over- and under-expression in MCF-7 cell-line. And these apoptosis-related genes closest to fully conserved cis-regulatory elements in network showed strong correlation with ER in MCF-7 cells. These results elucidate that ER regulates an evolutionarily conserved apoptosis pathway. This opens up new perspectives in the apoptosis of human breast cancer from the evolution of cis-regulatory elements. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.