Abstract Background: Breast cancer is the second leading cause of cancer-related deaths. Chemotherapy resistant breast cancer is poorly differentiated and displays aggressive clinical behavior. These tumors become resistant to cytotoxic agents and tumor relapse has been attributed to the presence of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Niclosamide has been shown to have broad clinical applications for the treatment of malignant tumors other than those caused by parasites. However, the specific functions and molecular mechanisms of niclosamide in chemo-resistant HER2 positive breast cancer are still unknown. Methods: HER2 positive breast cancer cell line, BT474, was continuously exposed to increasing concentrations of cisplatin (5-20 μM) to establish stable cell line resistant to cisplatin, BT-CR. Cell viability was determined by alamar blue. Apoptosis was determined by flow cytometry. Mammosphere formation assay was conducted to observe the self-renewal potential. Invasion ability was analyzed by transwell assay. Protein expression was determined by western-blot. Results: BT-CR had EMT and stem-like phenotype with higher invasion ability compared to naive sensitive cells. Alamar Blue assay showed combination of niclosamide with cisplatin could reverse cisplatin resistance. The combination index value of niclosamide combined with cisplatin was less than 1. Niclosamide in combination with cisplatin significantly enhanced apoptosis of BT-CR cells to over 50%. Western-blot showed niclosamide and cisplatin could reverse the EMT phenotype of BT-CR with E-cadherin up-regulated and N-cadherin and vimentin down-regulated. A significant reduction of Bcl-2 and Stat3 phosphorylation (Tyr705) levels was also confirmed. After treatment of niclosamide combined with cisplatin, the inhibition of mammosphere forming efficiency and capability of invasion was also observed in BT-CR. Conclusion: Our results revealed that niclosamide combined with cisplatin inhibited cell growth, invasion, EMT and stem-like phenotype in cisplatin resistant BT474 cells. The inhibitory effect of niclosamide was exerted by increasing apoptosis and down-regulated Bcl-2 expression, whilst inhibiting phosphorylation levels of STAT3. The results suggested that niclosamide combined with cisplatin appears to be a novel therapeutic way in chemo-resistant breast cancer. Citation Format: Junjun Liu. Niclosamide reverses cisplatin resistance by inhibiting Bcl-2 and Stat3 in HER2-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-33.