The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) inhibits DNA methyltransferase activity and sensitizes cancer cells to chemotherapy, but the mechanisms of its sensitization are not fully understood. Here, we show that 5-aza-CdR induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the human breast cancer MDA-231 cells. Induction of TRAIL by 5-aza-CdR correlated with inactivation of Akt. Furthermore, we show that overexpression of the active form of Akt by adenovirus infection or inhibition of the Akt downstream target glycogen synthase kinase 3 by its pharmacologic inhibitors abolishes TRAIL induction by 5-aza-CdR. Importantly, we show that the combined treatment of breast cancer cells with 5-aza-CdR and Adriamycin significantly increases apoptotic cell death compared with the treatment with either agent alone. Moreover, the combined treatment activated both death receptor and mitochondrial apoptotic pathways, whereas Adriamycin alone activated only the mitochondrial pathway while 5-aza-CdR failed to activate either. More importantly, down-regulation of TRAIL by small interference RNA silencing decreased 5-aza-CdR-mediated Adriamycin-induced caspase activation and apoptosis, thus conferring Adriamycin resistance. Taken together, our results suggest that induction of TRAIL by 5-aza-CdR is critical for enhancing chemosensitivity of breast cancer cells to Adriamycin.