The advent of combination therapy unprecedentedly shifted the paradigm of cancer treatment by reconstructing the conventional protocols. By identifying the anti-tumoral activity for different natural products, recent interest has focused on inventing the combined-modality strategies in order to increase the cure rates of cancer, while reducing the toxic side effects of current intensive regimens. To evaluate whether melatonin, the indolic hormone produced mainly by the pineal gland, could enhance the pro-apoptotic effect of arsenic trioxide (As₂O₃) in breast cancer, MCF-7 cells were treated with As₂O₃-plus-melatonin and then the survival, proliferative rate, caspase-3 activity, and mRNA expression level of anti-apoptosis target genes of NF-κB were investigated. Our results delineated that exposure of MCF-7 cells to As₂O₃ not only reduced the survival of the cells but also induced a caspase-3-dependent apoptotic cell death. Noteworthy, an enhanced induction of apoptosis was found using As₂O₃ in combination with melatonin. Moreover, RQ-PCR analysis revealed that the enhanced cytotoxic effect of As₂O₃ in the presence of melatonin is mediated, at least partly, through suppressing the expression of NF-κB anti-apoptotic target genes such as MCL-1, BCL-2, survivin, XIAP, and c-IAP1 in breast cancer cells. The resulting data showed that As₂O₃, either alone or in combination with melatonin, exerted significant cytotoxic effect against MCF-7 cells. However, further investigations are needed to provide valuable clues for expediting this combination as a therapeutic strategy for breast cancer.