Small GTPases play important roles in various aspects of cell division as well as membrane trafficking. We and others previously showed that ADP-ribosylation factor 6 (Arf6) is locally activated around the ingressing cleavage furrow and recruited to the Flemming body in late cytokinesis phases, and involved in faithful completion of cytokinesis. However, knockout of the Arf6 gene or Arf6 depletion by siRNAs did not drastically influence cytokinesis. We here show that, in addition to Arf6, Class I Arfs (Arf1 and Arf3) are localized to the Flemming body, and that double knockdown of Arf1 and Arf3 moderately increases the proportion of multinucleate cells and simultaneous knockdown of Arf1, Arf3 and Arf6 leads to severe cytokinesis defects. These observations indicate that Arf1 and Arf3 as well as Arf6 play important roles in cytokinesis. We further show that EFA6 (exchange factor for Arf6) activates not only Arf6 but also Arf1 in the cell. Taken together with our previous data, these Arf GTPases are likely to be locally activated by EFA6 and in turn targeted to the Flemming body to complete cytokinesis.