Pharmacological targeting of breast cancer stem cells (CSCs) is highly promising for the treatment of breast cancer, as the small population of CSCs appears responsible for tumor initiation and progression and also for resistance to conventional treatment. Here we report that the novel phosphosulindac (OXT-328, PS) selectively and effectively eliminates breast CSCs both in vitro and in vivo. PS reduced cell proliferation and induced apoptosis in various breast CSCs. Breast CSCs are resistant to conventional cancer drugs but are sensitive to PS. Long-term treatment of mixtures of cultured breast CSCs and breast cancer cells with PS preferentially eliminated the CSCs. PS impaired the ability of CSCs to form mammospheres and markedly suppressed the expression of CSC-related genes. More importantly, PS prevented by half (p = .06) the formation of tumors initiated by CSCs in immunodeficient mice, and inhibited by 83% (p < .05) the growth of already formed breast cancer xenografts, reducing the proportion of CSCs in them. PS suppressed the Wnt/β-catenin pathway by stimulating the degradation of β-catenin and its relocalization to the cell membrane and also blocked the epithelial-mesenchymal transition and the generation of breast CSCs. These results indicate that PS has a strong inhibitory effect against breast cancer, acting, at least in part, by targeting CSCs through a signaling mechanism involving Wnt signaling.