DNA methylation plays important roles in gene regulation and functions. Aberrant methylation, either hypomethylation or hypermethylation, has been reported to cause various diseases, especially cancers. Breast cancer ranked the fifth according to the number of cancer deaths in the world. To systematically characterize the epigenetic modification in breast cancer, we examined the genome-wide methylation profiling in 40 breast cancer cell lines. We identified a gene signature consisting of 345 differentially methylated genes, which could be used to discriminate estrogen receptor (ER)-negative and ER-positive breast cancer cell lines. This gene signature is promising for diagnosis and therapies of breast cancer. In the follow up functional analysis of this gene signature, three enriched networks could be highlighted. Interestingly, one of these networks contained estrogen receptor, implying its functional importance of ER-centric module. Finally, we examined the correlation between methylation and expression of these breast cancer cell lines. Very few genes showed significant correlation, suggesting that gene expression regulated by methylation is a complex biological process.