The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Forster resonance energy transfer-based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell's leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.