AbstractThe final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin‐like protein, Mitotic kinesin‐like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK‐4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly. Centralspindlin has several critical functions during cell division including positioning of the division plane, regulation of Rho family GTPases, as well as midbody assembly and abscission. In this review, we will examine the biochemistry of centralspindlin and its multiple functions during cell division. Remarkably, several of its critical functions are somewhat unexpected. Although endowed with motor domains, centralspindlin has an important role in generating stable, antiparallel microtubule bundles. Although it contains a Rho family GAP domain, it has a central role in the activation of RhoA during cytokinesis. Finally, centralspindlin functions as a motor protein complex, as a scaffold protein for key regulators of abscission and as a conventional RhoGAP. Because of these diverse functions, centralspindlin lies at the heart of the cytokinetic mechanism. © 2012 Wiley Periodicals, Inc