Vesicular structures called microvesicles (MVs) that are shed from the surfaces of cancer cells are capable of transferring oncogenic cargo to recipient cancer cells, as well as to normal cells, sending mitogenic signals that greatly enhance tumor growth. Because MVs are stable in the circulation, they also may have a key role in secondary colonization and metastasis. Thus, understanding how MVs are generated could have important consequences for interfering with cancer progression. Here we report that the small GTPase RhoA triggers a specific signaling pathway essential for MV biogenesis in various human cancer cells. Inhibiting the activity of different proteins comprising this pathway blocks MV biogenesis in the donor cancer cells and prevents oncogenic transformation in cell culture as well as tumor growth in mice. Although RhoA has often been implicated in human cancer, these findings now highlight a previously unappreciated role for this GTPase in malignant transformation, and demonstrate that blocking MV biogenesis may offer novel approaches for interfering with malignant transformation.