Abstract: Objective: To investigate the lethal effects of Parthenolide (PTL) on cancer stem cells (CSC) of mouse breast cancer in vitro. Methods:Serum-free mammosphere suspension culture fluid was used to culture4T1 cell lines in a manner designed to enrich for the breast cancer CSC. The 10th generation mammosphere cells were used as the experimental cells. The experimental cells were divided into5 groups: the control group, the 5-FU group, the PTL group, the 5-FU+PTL group and the PTL+NAC (N-Acetylcysteine) group. The experimental cells were treated with the appropriate drugs, and then the cells were cultured using serum-free medium. Seven days later, the mammosphere forming conditions were observed and mammosphere cells were collected for testing the CD44+ CD24-/low cell content, detecting the non-fluorescent dye staining side population (SP) cells ratio, and evaluating the MDR 1, BCRP and mRNA expression levels. Results:The CSC in the 4T1 cell line were enriched by the serum-free suspension culture, and the CD44+CD24-/low cell ratio reached 68.9 ± 3.78% . After treatment with different drugs, the cells of the control, the5-FU and the PTL + NAC groups formed visible mammospheres. Nevertheless, fewer mammospheres were formed in the cells of the PTL group, and no cells survived in the 5-FU+PTL group. In the control, 5-FU, PTL, and PTL+NAC groups, the CD 44+CD24-/low cell contents were 71.2 ± 2.3%,75.6 ± 3.1%,10.3 ± 1.9% and 58.1 ± 2.6% respectively, and the percentage of the non-fluorescent staining (SP) cells were 56.7 ± 3.4% ,62.0 ± 2.7% ,8.1 ± 1.1% and 51.5 ± 2.3% , respectively. The cell contents were more significantly decreased in the PTL group than in the other three groups (P<0.01), with a statistical significance when comparing the differences among the groups (P<0.01). Compared with the cells of the control group, the relative expression of MDR1 and BCRP genes was 1.16± 0.21and 1.09± 0.13in the cells of the 5-FU group, respectively; 0.22± 0.15 and 0.27± 0.11in the cells of the PTL group, respectively; and 0.89± 0.18and 0.93± 0.14in the cells of the PTL+NAC group, respectively. Conclusion : PTL can target the CSC of breast cancer, change the state of CSC, and significantly reduce the CSC content of the cultured mammosphere cells. The antioxidant NAC can apparently antagonize this effect of PTL in vitro.