RhoGTPases family are involved in several biological process including gene transcription, cell polarity, migration and invasion. RhoGTPases switch between on and off states and they are regulated by several GEFs (activators) and GAPs/RhoGDIs (inactivators). The aim of this work is to study the role of a particular RhoA-GEF, GEF-H1, in breast cancer (BC) progression. We observed by immunostaining a significant increase of GEF-H1 protein expression in BC human biopsies compared with non-tumoral tissue. In addition, we observed that GEF-H1 expression correlates with the invasive potential of human and murine BC cell lines. To further study the role of GEF-H1 in tumor development, we generated GEF-H1-knock out (KO) BC cells using CRISPR/Cas9 technology. We observed a decreased in proliferation, migration, invasion and anchorage-independent colony formation in GEF-H1-KO cells versus wild type (WT) cells. These results correlate with a reduced focal adhesion formation and signalling. Furthermore, BALB/c mice were subcutaneously inoculated with GEF-H1 KO cells, showing a significant delay in tumor formation and lung metastasis development compared with WT cells. These results showed that GEF-H1-RhoA activation may mediate the signalling involved in controlling cell proliferation, migration and invasion of BC cells. In vivo assays and human biopsy analyses suggest that GEF-H1 expression in BC cell might indeed contribute to tumor progression.