A subset of primary breast cancers and breast cancer cell lines express a hypermethylation defect characterized by DNMT hyperactivity and DNMT3b overexpression. The objectives of this project were (i) to determine if targeting the methylome enhances the sensitivity of breast cancer cells to chemotherapy, and (ii) to elucidate the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer. To address the first objective, hypermethylator breast cancer cell lines were treated with demethylating agent (5-aza-2'-deoxycytidine) and/or were subjected to RNAi mediated DNMT3b knockdown (KD), and then tested for sensitivity to doxorubicin hydrochloride, paclitaxel, and 5-fluorouracil. The results show that pharmacologic demethylating pretreatment sensitizes hypermethylator breast cancer cells to cell killing by cytotoxic drugs, and provide proof of concept that direct targeting of DNMT3b also improves cell kill by these drugs. These findings suggest that targeting the methylome improves chemotherapeutic efficacy of cytotoxic drugs against hypermethylator breast cancer cells as a function of dose and duration of exposure to demethylating treatment. To address the second objective, the expression of microRNAs (miRs) that regulate or are predicted to regulate DNMT3b were examined in hypermethylator or non-hypermethylator breast cancer cell lines and in primary breast cancers. Hypermethylator cell lines express diminished levels of regulatory miRs compared to non-hypermethylator cell lines. Mechanistic studies were conducted to establish the role of miR expression in the hypermethylation defect. Antagomir-mediated knockdown of regulatory miRs in nonhypermethylator cell lines resulted in increased DNMT3b mRNA and forced reexpression of regulatory miRs reduced DNMT3b mRNA in hypermethylator cell lines. In primary breast cancers, miR expression patterns revealed two distinct subsets among the basal-like subtype. Most hypermethylator basal like cancers exhibit diminished expression of regulatory miRs. These findings strongly suggest that diminished expression of multiple regulatory miRs contributes to DNMT3b overexpression. Together, these results support the conclusion that the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer cells is the loss of post-transcriptional regulation of DNMT3b by regulatory miRs, and that combined epigenetic and cytotoxic treatment will improve the efficacy of breast cancer chemotherapy.