Whereas accumulating studies have supported the cancer stem cell theory, a specific therapy targeting a cancer stem cell subpopulation has not been established. Here, we show that dual-specificity tyrosine phosphorylation-kinase 2 (DYRK2) is a novel negative regulator for formation of breast cancer stem cells. Downregulation of DYRK2 promotes cancer stem-like traits in vitro, tumourigenesis in vivo and the proportion of the cancer stem cell population in human breast cancer tissues. We found that Krupple-like factor 4 (KLF4) serves as a key mediator of DYRK2's control over the cancer stem phenotype. Reduced DYRK2 expression increases KLF4 expression, which induces cancer stem-like properties. We identified androgen receptor (AR) as a transcription factor binding to the KLF4 promoter region; this process is dependent on DYRK2 kinase activity. Our findings delineate a mechanism of cancer stem cell regulation by the DYRK2-AR-KLF4 axis in breast cancer. Targeting of this pathway may be a promising strategy against breast cancer stem cells.