UNLABELLEDTargeted therapy against triple-negative breast cancers, which lack expression of the estrogen, progesterone, and HER2 receptors, is not available and the overall response to cytotoxic chemotherapy is poor. One of the molecular hallmarks of triple-negative breast cancers is increased expression of genes that are transcriptionally activated by hypoxia-inducible factors (HIFs), which are implicated in many critical aspects of cancer progression including metabolism, angiogenesis, invasion, metastasis, and stem cell maintenance. Ganetespib is a second-generation inhibitor of heat shock protein 90 (HSP90), a molecular chaperone that is essential for the stability and function of multiple client proteins in cancer cells including HIF-1α. In this study, human MDA-MB-231 and MDA-MB-435 triple-negative breast cancer cells were injected into the mammary fat pad of immunodeficient mice that received weekly intravenous injections of ganetespib or vehicle following the development of palpable tumors. Ganetespib treatment markedly impaired primary tumor growth and vascularization, and eliminated local tissue invasion and distant metastasis to regional lymph nodes and lungs. Ganetespib treatment also significantly reduced the number of Aldefluor-positive cancer stem cells in the primary tumor. Primary tumors of ganetespib-treated mice had significantly reduced levels of HIF-1α (but not HIF-2α) protein and of HIF-1 target gene mRNAs encoding proteins that play key roles in angiogenesis, metabolism, invasion, and metastasis, thereby providing a molecular basis for observed effects of the drug on the growth and metastasis of triple-negative breast cancer.KEY MESSAGESTriple-negative breast cancers (TNBCs) respond poorly to available chemotherapy. TNBCs overexpress genes regulated by hypoxia-inducible factors (HIFs). Ganetespib induces degradation of HSP90 client proteins, including HIF-1α. Ganetespib inhibited TNBC orthotopic tumor growth, invasion, and metastasis. Ganetespib inhibited expression of HIF-1 target genes involved in TNBC progression.