Despite all the advances in research leading to improved therapies, resistance to current anti-cancer therapy remains a critical clinical problem. ER-positive breast cancer risk of recurrence after stopping tamoxifen treatment can be up to 41% in some cases, highlighting the need for additional approaches to reduce this risk. Increased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in breast cancer. SOX2 has potential as a biomarker of resistance to treatment and as a therapeutic target, but targeting transcription factors is challenging. We have identified an inhibitor that specifically blocks in vitro binding to endogenous SOX2, leading to impaired tumour formation by reducing CSC content in a SOX2-dependent manner and stem cell depletion in vivo. These findings suggest that combination therapies that include SOX2 inhibition could be useful to treat tamoxifen-resistant breast cancer.