Inhibition of motor protein activity has been linked with defects in the formation of poles in the spindle of dividing cells. However, the molecular mechanisms underlying the functional relationship between motor activity and centrosome dynamics have remained uncharacterised. Here, we characterise KIFC5A, a mouse kinesin-like protein that is highly expressed in dividing cells and tissues, and is subject to developmental and cell-type-specific regulation. KIFC5A is a minus-end-directed, microtubule-dependent motor that produces velocities of up to 1.26 microm minute(-1) in gliding assays and possesses microtubule bundling activity. It is nuclear in interphase, localises to the centre of the two microtubule asters at the beginning of mitosis, and to spindle microtubules in later mitotic phases. Overexpression of KIFC5A in mouse cells causes the formation of aberrant, non-separated microtubule asters and mitotic arrest in a prometaphase-like state. KIFC5A knockdown partly rescues the phenotype caused by inhibition of plus-end-directed motor Eg5 by monastrol on the mitotic spindle, indicating that it is involved in the balance of forces determining bipolar spindle assembly and integrity. Silencing of KIFC5A also results in centrosome amplification detectable throughout the cell cycle. Supernumerary centrosomes arise primarily as a result of reduplication and partly as a result of cytokinesis defects. They contain duplicated centrioles and have the ability to organise microtubule asters, resulting in the formation of multipolar spindles. We show that KIFC5A interacts with nucleotide-binding proteins 1 and 2 (Nubp1 and Nubp2), which have extensive sequence similarity to prokaryotic division-site-determining protein MinD. Nubp1 and Nubp2 also interact with each other. Knockdown of Nubp1 or double knockdown of Nubp1 and Nubp2 (Nubp1&Nubp2) both phenocopy the KIFC5A silencing effect. These results implicate KIFC5A and the Nubp proteins in a common regulatory pathway involved in the control of centrosome duplication in mammalian cells.