Human Ninein (hNinein) is implicated in centrosomal microtubule nucleation and microtubule anchoring in interphase cells and may act as a scaffold protein, but its direct interaction partners remain unexplored in the centrosome. In this report, we show clearly that a spindle-associated protein, Astrin, interacts and co-localizes with hNinein at the centrosome during the S and G2 phases, and this complex may dissociate in the M phase. We also demonstrate that the truncated forms of hNinein, which could interfere with gamma-tubulin and function as dominant-negative mutants, are able to affect Astrin localization to the centrosome. Moreover, siRNA-mediated knockdown of hNinein in HeLa cells causes Astrin to fail to target to the centrosome, whereas hNinein can localize at the centrosome in the absence of Astrin. In addition, reduction in hNinein protein levels causes mislocalization of Astrin with the spindle apparatus and results in the formation of an aberrant mitotic spindle. Collectively, these data suggest that hNinein is required for targeting Astrin to the centrosome during the S and G2 phases. We therefore propose a model wherein hNinein regulates the dynamic movement of Astrin throughout the cell cycle and this interaction, in turn, is required for maintenance of centrosome/spindle pole integrity.