Cancer stem cells (CSCs) have been shown to be resistant to current anticancer therapies and the induction of oxidative stress is an important mechanism of action for many anticancer agents. However, it is still largely unknown how CSCs respond to hydrogen peroxide (H2O2)-induced oxidative stress. Here, we show that the levels of reactive oxygen species (ROS) are markedly lower in breast CSCs (BCSCs) than that in non-cancer stem cells (NCSCs). A transient exposure of breast cancer cells to sublethal doses of H2O2 resulted in a dose-dependent increase of the epithelium-specific antigen (ESA)(+)/CD44(+)/CO24(-) subpopulations, a known phenotype for BCSCs. Although BCSCs survived sublethal doses of H2O2 treatment, they lost the ability to form tumor spheres and failed to generate colonies as demonstrated by mammosphere-formation and clonogenic assays, respectively. Mechanistic studies revealed that H2O2 treatment led to a marked increase of senescence-associated beta-galactosidase activity but only minimal apoptotic cell death in BCSCs. Furthermore, H2O2 triggers p53 activation and promotes p21 expression, indicating a role for the p53/p21 signaling pathway in oxidative stress-induced senescence in BCSCs. Taken together, these results demonstrate that the maintenance of a lower level of ROS is critical for CSCs to avoid oxidative stress and H2O2-induced BCSC loss of function is likely attributable to oxidative stress triggered senescence induction, suggesting that ROS-generating drugs may have the therapeutic potential to eradicate drug-resistant CSCs via induction of premature senescence. (C) 2019 Elsevier Inc. All rights reserved.