Cell division requires coordinated regulation of chromosome segregation and cytokinesis. Although much is known about the function of the protease separase in promoting sister chromosome separation, the role of separase during cytokinesis is unclear. We show that separase localizes to the ingressing furrow and midbody during cytokinesis in the C. elegans embryo. Loss of separase function during the early mitotic divisions causes cytokinesis failure that is not due to eggshell defects or chromosome nondisjunction. Moreover, depletion of separase causes the accumulation of RAB-11-positive vesicles at the cleavage furrow and midbody that is not a consequence of chromosome nondisjunction, but is mimicked by depletion of vesicle fusion machinery. Collectively, these data indicate that separase is required for cytokinesis by regulating the incorporation of RAB-11-positive vesicles into the plasma membrane at the cleavage furrow and midbody.