AbstractHypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.