Abstract Background: Salinomycin has recently proven to be highly effective against a model of breast cancer stem cells in vitro and in vivo. Moreover, treatment with this compound induced epithelial differentiation of tumor cells. A population of Brain Tumor Stem Cells (BTSCs) has been identified in malignant brain tumors. This population of cells has been found responsible for the initiation of these malignancies and may constitute the cause of resistance of malignant brain tumors to chemotherapy and radiotherapy and therefore responsible for the lethal recurrence after surgery. We hypothesize that salinomycin could be successfully implemented for the treatment of brain tumors and specifically for the eradication of BTSCs. Material and Methods: In vitro cytotoxic effect of salinomycin or TMZ was assessed in a panel of BTSCs lines (n=11), established glioma cell lines (n=4) and normal human astrocytes (NHA) by MTT. Self-renewal was assayed by single cell plating and limited dilution assay. Cell cycle was evaluated by flow cytometry using PI staining. In addition, we performed QT-RT-PCR and western blot analysis to assess the expression of different stem cells markers before and after the treatment. In addition, global gene expression analysis was performed using an affymetrix array. Survival studies in animal models are in progress. Results: Our results showed that salinomycin presents a potent antitumoral effect against BTSCs and glioma cell lines. Importantly, salinomycin displayed an IC50 ranging from 100 to 10000 folds lower than TMZ (currently the gold standard treatment for malignant brain tumors) in the same cell lines. Interestingly, salinomycin reduced the number of self-renewal cells in treated-BTSCs. This was confirmed with the decreased in the expression of stem cell markers such as Sox2 and Bmi1. Cell cycle analysis showed that unlike TMZ treated cells, which arrest in G2, salinomycin treated cells did not experienced growth arrest and progress through the cell cycle to end up dying. Conclusions: Altogether our data show that salinomycin is more potent than temozolomide for the treatment of brain tumor stem cells and therefore could be a promising agent against malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3340.