Rho GTPases are involved in several biological and pathological processes, including gene transcription, cell polarity, migration and invasion. Rho guanine nucleotide exchange factor-H1 (GEF-H1) is the unique RhoA activator that binds to microtubules and its localization and activity is regulated in part by fibronectin-binding integrins. The aim of this work is to investigate the role of GEF-H1 in breast cancer progression. We report that the expression of GEF-H1 is higher in human breast cancer biopsies than in normal tissue. Furthermore, the expression of GEF-H1 is higher in breast cancer cell lines than in non-tumoral cell lines. We observed a significant reduction in the number of focal adhesions, stress fibers formation and altered downstream signaling in GEF-H1 knockout (KO) breast cancer cell lines, resulting in decreased cell proliferation, migration, adhesion and invasion. In addition, orthotopic implantation of GEF-H1 KO cells into mammary fat pads of Balb/c mice showed a significant delay in tumor formation and lung metastasis development compared to control breast cancer cells. These results suggest that activation of GEF-H1/RhoA mediates cytoskeletal remodeling and signaling pathways involved in cell proliferation, migration, and invasion of breast cancer cells. In vivo assays and human biopsy studies suggest that GEF-H1 expression indeed contributes to breast tumor progression and could be postulated as a potential biomarker.