Two subtypes of the estrogen receptor (ER), ERα and ERβ, mediate the actions of estrogens, and although 70% of human breast cancers express ERβ along with ERα, little is known about the possible comodulatory effects of these two ERs. To investigate this, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing different levels of ERβ, along with their endogenous ERα, and have examined the effects of ERβ and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ERβ had diverse effects on gene expression, enhancing or counteracting ERα regulation for distinct subsets of estrogen target genes. Strikingly, ERβ in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that were normally only regulated by ERα with E2. In addition, ERβ plus E2 elicited the expression of a unique group of genes that were not regulated by ERα plus E2 alone. The expression of genes in many functional categories were modulated by ERβ, with the greatest numbers associated with transcription factors and signal transduction pathways. Regulation of multiple components in the TGFβ and semaphorin pathways, and of genes controlling cell cycle progression and apoptosis, may contribute to the suppression of cell proliferation observed with ERβ. Our observations suggest that the relative levels of ERβ and ERα in breast cancers are likely to impact cell proliferation and the activities of diverse signaling pathways and their response to ER ligands and endocrine therapies.