The sub-population of tumor cells termed ‘cancer stem cells’ (CSCs) possess the capability to generate tumors, undergo epithelial–mesenchymal transition (EMT) and are implicated in metastasis, making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here, we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines, or in primary metastatic breast cancer cells, results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators, as well as drivers of ‘stemness’, such as Notch1 and Jagged1 in isolated CSCs. In addition, treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore, combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and ‘stemness’ phenotypes of these cells, making CAIX an important therapeutic target for selectively depleting breast CSCs.