Abstract Tumor initiating cells or cancer stem cells (CSCs) were characterized as a unique subpopulation with stem cell features in many types of cancer. Although there has been a number of studies on breast cancer stem cells, inhibitory mechanisms of tumor initiation, progression, and resistance to therapy are unknown. Salinomycin is a chemical isolated from Streptomyces albus and a potassium ionophore. It has been used for many years to control coccidiosis. Recently, salinomycin has been reported to act as selective breast cancer stem cell inhibitor because of its property of reducing the cancer stem cell and inducing the apoptosis in cancer cell. Thus, we investigated the anti-cancer property of salinomycin in mouse mammary epithelial cell. With CDμgeo (mouse mammary epithelial cell), MTT assay was used to confirm cell viability after salinomycin treatment (0, 0.5, 1, 2, 5, 10 μm/ml) upon anchorage-dependent state. We observed that salinomycin reduced the cell proliferation dose-dependently. Also, mammosphere formation assay was performed to observe stem cell-like property after salinomycin treatment in anchorage-independent state. We confirmed salinomycin reduced the number and the size of mammosphere in 72, 144, and 216 hours. We performed salinomycin pre-treatment (0, 0.5, 1, 2, 5 μm/ml) for 12-48 hours upon anchorage-dependent state. Results by western blotting revealed that salinomycin promoted apoptosis of mouse mammary epithelial cells by activating both caspase-3 and -9 and inducing PARP cleavage. To confirm the reduction of the side population level, we performed FACS analysis using CD49+, CD29+, and CD24+ markers. Based on our findings, we concluded that anti-cancer effect of salinomycin reduced stemness and induced apoptosis in mouse mammary epithelial cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3359. doi:1538-7445.AM2012-3359