Tastin was previously characterized as an accessory protein for cell adhesion that participates in early embryo implantation. Here, we report that tastin is also required for spindle assembly during mitosis. Tastin protein levels peaked in the G(2)/M phase and abruptly declined after cell division. Microscopy showed that tastin is primarily localized on the microtubules, centrosomes, and the mitotic spindle during the cell cycle. Tastin interacted with the dynein intermediate chain, p150(Glued), and gamma-tubulin in addition to Tctex-1 ( the light chain of dynein). Overexpression of tastin led to monopolar spindle formation, whereas loss of tastin expression caused profound mitotic block and preferentially induced multipolar spindles. These multipolar spindles were generated through a loss of cohesion in mitotic centrosomes; specifically, tastin depletion caused the fragmentation of pericentrosomal material and the splitting of the centrioles at the spindle poles. Tastin depletion induced centrosome abnormalities exclusively during mitosis and required both microtubule integrity and Eg5 activity. However, tastin depletion did not disrupt the organization of spindle poles, as revealed by localization of nuclear mitotic apparatus protein (NuMA) and the p150(Glued) component of dynactin. These data indicate that the major function of tastin during mitosis is to maintain the structural and dynamic features of centrosomes, thereby contributing to spindle bipolarity.