During cancer development, coordinated changes in cell motility and cell cycle progression are required for the gradual transformation of normal cells into cancer cells. Previous studies have shown that ARF6 is a critical regulator of epithelial cell integrity and motility via its role in membrane movement and actin-based cytoskeletal remodeling. Recently, we have found that ARF6 also plays a role during cell division. It localizes to the cleavage furrow and midbody of cells during mitosis, and its activity is regulated during cytokinesis. Here, we investigate the requirement for ARF6 during mitosis and find that depletion of ARF6 using RNA interference disrupts the completion of cytokinesis. This finding demonstrates that ARF6 is essential during the final stages of cytokinesis. In addition, we have identified Ku70, a DNA-binding protein that is required for DNA damage repair, as a new ARF6-interacting protein and found that it is part of a complex with ARF6, especially during mitosis. These results clarify the importance of ARF6 activity during cytokinesis and begin to reveal other molecules that may contribute to the function of ARF6.