BACKGROUND: Breast cancer is the most common malignant tumor in women. Due to limited treatment outcome and high rate of metastasis, the prognosis is especially poor for triple-negative breast cancer. It is urgent to discover and develop novel agents for treatment of breast cancer. Herein, we investigated the potential mechanisms of Oleandrin's (a cardiac glycoside) cytotoxic activity against breast cancer cells. METHODS: Cell proliferation was assessed by xCELLigence Real-Time Cell Analyzer (RTCA)-MP system. Apoptotic cells were detected by using Annexin V/PI staining and nuclear fragments observation. The effect of oleandrin on ATP1B3 expression and markers of ER stress were determined by western blot. A primary cell sensitivity assay was performed via a collagen gel droplet-embedded culture drug sensitivity method (CD-DST). RESULTS: Oleandrin suppressed cell proliferation and colony formation in the three breast cancer cell lines but did not affect normal mammary epithelial cells. Additionally, the expression of ATP1B3 was higher in the three breast cancer cell lines compared to MCF10A cells. Treatment with oleandrin increased the number of apoptotic cells and led to nuclear pyknosis, fragmentation, and apoptotic body formation in breast cancer cells. Furthermore, oleandrin treatment increased expression of Bax and Bim but decreased that of Bcl-2. Treatment with oleandrin also upregulated the expression of endoplasmic reticulum stress associated proteins, including eIF2α, ATF4, and CHOP, but not PERK. oleandrin treatment also induced the phosphorylation of PERK and eIF2α. Of note, oleandrin exhibited antitumor effects on patient-derived breast cancer cells under three-dimensional culture conditions. CONCLUSIONS: Taken together, our results suggest that oleandrin induces mitochondrial-mediated apoptosis by activating endoplasmic reticulum stress in breast cancer. Moreover, oleandrin may be an effective strategy for the treatment of breast cancer.