Radiation is widely used for cancer treatment but the radioresistance properties of cancer stem cells (CSCs) pose a significant challenge to the success of cancer therapy. Nuclear factor erythroid-2-related factor 2 (Nrf2) has emerged as a prominent regulator of cellular antioxidant responses and its over-activation is associated with drug resistant in cancer cells. However, the role of Nrf2 signaling in regulating the response of CSCs to irradiation has yet to be defined. Here, we show that exposure of triple-negative breast cancer (TNBC) cells to ionizing radiation (IR) upregulates Nrf2 expression and promotes its nuclear translocation in a reactive oxygen species (ROS)-dependent manner. Ectopic overexpression of Nrf2 attenuates, whereas knockdown of Nrf2 potentiates IR-induced killing of TNBC CSCs. Mechanistically, we found that Nrf2 knockdown increases IR-induced ROS production and impedes DNA repair at least in part via inhibition of DNA-PK. Furthermore, activation of Nrf2 by sulforaphane diminishes, whereas inhibition of Nrf2 by ML385 enhances IR-induced killing of TNBC CSCs. Collectively, these results demonstrate that IR-induced ROS production can activate Nrf2 signaling, which in turn counteracts the killing effect of irradiation. Therefore, pharmacological inhibition of IR-induced Nrf2 activation by ML385 could be a new therapeutic approach to sensitize therapy-resistant CSCs to radiotherapy.Keywords: Breast cancer stem cells; DNA repair; DNA-PK; Ionizing radiation; Nrf2; Reactive oxygen species.