EVI5 has been shown to be a novel centrosomal protein in interphase cells. In this report, we demonstrate using immunofluorescence microscopy that EVI5 has a dynamic distribution during mitosis, being associated with the mitotic spindle through anaphase and remaining within the midzone and midbody until completion of cytokinesis. Knockdown of EVI5 using siRNA results in a multinucleate phenotype, which is consistent with an essential role for this protein in the completion of cytokinesis. The EVI5 protein also undergoes posttranslational modifications during the cell cycle, which involve phosphorylation in early mitosis and proteolytic cleavage during late mitosis and cytokinesis. Since the subcellular distribution of the EVI5 protein was similar to that characteristic of chromosomal passenger proteins during the terminal stages of cytokinesis, we used immunoprecipitation and GST pull-down approaches to demonstrate that EVI5 is associated with the aurora B kinase protein complex (INCENP, aurora B kinase and survivin). Together, these data provide evidence that EVI5 is an essential component of the protein machinery facilitating the final stages of cell septation at the end of mitosis.