Accumulating evidence suggests that B-catenin signaling in breast cancer stem cells (CSCs) is closely correlated to chemoresistance and adenosine triphosphate (ATP)-binding cassette subfamily G2 (ABCG2) expression. Targeting the aberrant beta-catenin signaling in CSCs has become a promising strategy to improve chemosensitivity in cancer treatment. In a pilot screening study, we found that the natural compound isoliquiritigenin (ISL) blocked B-catenin transcription activity with the highest inhibition ratio. Here, we investigated the chemosensitizing effects of ISL on breast CSCs and the underlying mechanisms regulating the beta-catenin pathway. ISL could have synergistic effects with chemotherapeutic drugs to inhibit breast cancer cell proliferation and colony formation. In addition, ISL could significantly limit the side population and CSC ratios in breast cancer cells, accompanied by inhibited self-renewal and multidifferentiation abilities. A mechanistic study revealed that ISL could inhibit beta-catenin/ABCG2 signaling by activating the proteasome degradation pathway. The drug affinity responsive target stability strategy further identified GRP78 as the direct target of ISL. Subsequent molecular docking analysis and functional studies demonstrated that ISL could dock into the ATP domain of GRP78 and thereby inhibit its ATPase activity, resulting in its dissociation from beta-catenin. An in vivo study also suggested that ISL could chemosensitize breast CSCs via the GRP78/beta-catenin/ABCG2 pathway, with little toxicity in normal tissues and mammary stem cells. Taken together, the data from this study not only suggest ISL as a natural candidate to enhance breast CSC chemosensitivity but also highlight the significance of GRP78 in mediating cancer drug resistance and beta-catenin signaling in CSCs.