Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that Rac1b, the only known alternatively spliced variant of the small GTPase Rac1, is expressed in a subset of BCSCs in vivo and its function is required for the BCSC maintenance and the chemoresistance of breast tumor cells. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers BCSC activity in vivo and increases the chemosensitivity of primary tumor cells to doxorubicin. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that will improve the effectiveness of currently available chemotherapy modalities.