AbstractThe Arf6-specific exchange factor EFA6 is involved in the endocytic/recycling pathway for different cargos. In addition EFA6 acts as a powerful actin cytoskeleton organizer, a function required for its role in the establishment of the epithelial cell polarity and in neuronal morphogenesis. We previously showed that the C-terminus of EFA6 (EFA6-Ct) is the main domain which contributes to actin reorganization. Here, by in vitro and in vivo experiments, we sought to decipher, at the molecular level, how EFA6 controls the dynamic and structuring of actin filaments. We showed that EFA6-Ct interferes with actin polymerization by interacting with and capping actin filament barbed ends. Further, in the presence of actin mono-filaments, the addition of EFA6-Ct triggered the formation of actin bundles. In cells, when the EFA6-Ct was directed to the plasma membrane, as is the case for the full-length protein, its expression induced the formation of membrane protrusions enriched in actin cables. Collectively our data explain, at least in part, how EFA6 plays an essential role in actin organization by interacting with and bundling F-actin.