Origin recognition complex (ORC) proteins serve as a landing pad for the assembly of a multiprotein prereplicative complex, which is required to initiate DNA replication. During mitosis, the smallest subunit of human ORC, Orc6, localizes to kinetochores and to a reticular-like structure around the cell periphery. As chromosomes segregate during anaphase, the reticular structures align along the plane of cell division and some Orc6 localizes to the midbody before cells separate. Silencing of Orc6 expression by small interfering RNA (siRNA) resulted in cells with multipolar spindles, aberrant mitosis, formation of multinucleated cells, and decreased DNA replication. Prolonged periods of Orc6 depletion caused a decrease in cell proliferation and increased cell death. These results implicate Orc6 as an essential gene that coordinates chromosome replication and segregation with cytokinesis.