Abstract Drug repositioning can benefit from reduced development risk in terms of safety, toxicity and dosage, as it centers upon drugs that have already been tested in clinical trials. Disulfiram (DSF), a drug for the treatment of alcoholism, has potential anti-cancer activities in a copper (Cu)-dependent manner. In the present study, we investigated the effect of DSF on cell viability, apoptosis, breast cancer stem cells (BCSC)-like properties and p53 activation in ER-positive breast cancer MCF7 and T47D cells. DSF treatment caused a significant suppression of cell viability and marked induction of cell death in a Cu-dependent manner. This response was accompanied by caspase-3/-7 and p53 activation in ER-positive breast cancer cells. The latter phenomenon was associated with G1 phase arrest, coinciding with a significant increase in accumulation of nuclear p21 and downregulation of cyclin D1 and survivin. DSF treatment elicits a significant inhibitory effect on these BCSC-like properties including ALDH1 activity and CD44+/CD24- subpopulations. To examine the effect of DSF on mammosphere-forming ability, MCF7 and T47D cells were cultured in anchorage-independent serum-free culture conditions in the presence or absence of DSF and Cu. DSF considerably suppressed mammosphere formation, as evidenced by significant reduction in the number and volume of mammospheres. Our results demonstrate that DSF induces apoptosis and effectively targets cancer stem-like population, suggesting that disulfiram may be potentially effective for the treatment of ER-positive cancer. Citation Format: Seojin Jang, Eunhye Oh, Yoon-Jae Kim, Tae-Min Cho, Jung Min Park, Soeun Park, Minsu Park, Jae Hong Seo, Ji Young Kim. Disulfiram targets both proliferating cancer cells and cancer stem-like population in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1152.