Transcriptional effects of estrogen result from its activation of two estrogen receptor (ER) isoforms; ERα that drives proliferation and ERβ that is antiproliferative. Expression of ERβ in xenograft tumors from the T47D breast cancer cell line reduces tumor growth and angiogenesis. If ERβ can halt tumor growth, its introduction into cancers may be a novel therapeutic approach to the treatment of estrogen-responsive cancers. To assess the complete impact of ERβ on transcription, we have made a full transcriptome analysis of ERα-and ERβ-mediated gene regulation in T47D cell line with Tet-Off regulated ERβ expression. Of the 35 000 genes and transcripts analysed, 4.1%(1434) were altered by ERα activation. Tet withdrawal and subsequent ERβ expression inhibited the ERα regulation of 998 genes and, in addition, altered expression of 152 non-ERα-regulated genes. ERα-induced and ERβ-repressed genes were involved in proliferation, steroid/xenobiotic metabolism and ion transport. The ERβ repressive effect was further confirmed by proliferation assays, where ERβ was shown to completely oppose the ERα–E2 induced proliferation. Additional analysis of ERβ with a mutated DNA-binding domain revealed that this mutant, at least for a quantity of genes, antagonizes ERα even more strongly than ERβ wt. From an examination of the genes regulated by ERα and ERβ, we suggest that introduction of ERβ may be an alternative therapeutic approach to the treatment of certain cancers.