Estradiol (E2) is believed to modulate physiological functions relevant to osteoblast biology through the actions of estrogen receptors (ER) that in turn regulate the expression of target genes. The molecular effects of estrogen action in bone remain to be fully elucidated. This study reports a genome-wide molecular and computational analysis of the interaction between estrogen receptor (ER) and regulatory elements on the DNA of target genes in human primary osteoblasts. Of approximately 54000 gene probes surveyed in this study, a total of 375 genes were upregulated and 418 genes were downregulated on exposure to estradiol, with only 46 of these being direct target genes after 24 h, as determined by concomitant cycloheximide treatment. Computational analysis discovered several pathways where E2 co-regulates multiple functionally linked components. Examination of the genomic sequence of insulin growth factor binding protein 4 (IGFBP4) located ER response elements within the first intron. Using chromatin immunoprecipitation (ChIP) we show a site-and cell-specific recruitment of transcription factors to this newly identified regulatory region. Transient transfection studies revealed that this intronic region acts as a functional promoter in human osteoblasts. Taken together, this analysis provides a comprehensive gene transcription profile and identifies several genes of potential physiological importance in controlling estrogen mediated signaling in primary osteoblasts.