Endometrioid uterine cancers are known to develop from hypertrophic endometrium supported by increased estrogen concentrations.Recent studies indicate that DNA hypermethylation with the resulting expression silence of DNA repair genes and tumor suppressor genes plays a critical role in endometrial carcinogenesis.The cause of aberrant DNA methylation, however, is unknown.We previously showed that DNA methyltransferases (DNMT) are overexpressed in endometrioid carcinoma.In this study we characterize a novel pathway by which estrogen induces DNA methylation.We show that estrogen upregulates both DNMT3B transcription and de novo methylation activities in ER-positive endometrial and breast cancer cells.This effect is blocked by the estrogen antagonist but enhanced by ER overexpression, indicating an estrogen-specific, ER-dependent action.We demonstrate that estrogen treatment result in significant changes in the re-methylation dynamics of PEG3 tumor suppressor gene following DNMT inhibitor-mediated de-methylation.Moreover, we detected a significant DNA methylation alteration in the tumor suppressor gene Runx3 following ER overexpression.These results have provided evidence for a functional interaction between estrogenic and epigenetic pathways.The study reveals a potential molecular mechanism involved in the development of aberrant DNA hypermethylation and consequently, pathogenesis of estrogen-induced endometrial cancers.