Melanoma cell migration across vascular endothelial cells is an essential step of tumor metastasis. Here, we provide evidence that RacGAP1, a cytokinesis-related Rho GTPase-activating protein, contributed to this process. Depletion of RacGAP1 with RacGAP1-targeting siRNA or overexpression of RacGAP1 mutant (T249A) attenuated melanoma cell transendothelial migration and concomitant changes of adherens junctions. In addition, RacGAP1 promoted the activations of RhoA, FAK, paxillin and triggered focal adhesion formation and cytoskeletal rearrangement. By overexpressing FAK-related non-kinase (FRNK) in endothelium, we showed that RacGAP1 mediated endothelial barrier function loss and melanoma transmigration in a focal adhesion-dependent manner. These results suggest that endothelial RacGAP1 may play critical roles in pathogenic processes of cancer by regulating endothelial permeability. (C) 2014 Elsevier Inc. All rights reserved.