Abstract The microtubule cytoskeleton orchestrates the cellular dynamics and plasticity that underlies cellular morphogenesis. Microtubule binding proteins known as “TIP” proteins regulate the dynamic properties of microtubules during chromosomal segregation and cellular division. Our recent study revealed that TIP150 is a novel plus end tracking protein interacting with EB1 and MCAK in vitro and in vivo (EMBO Reports. 2009. 10:857-65). However, it is unclear whether and how this interaction regulates chromosome segregation in mitosis. We hypothesize that Tip150 is essential for chromosome segregation, and governs proper kinetochore-microtubule attachment at the microtubule plus end. To delineate the molecular function of TIP150 and its regulation in chromosome segregation and the mitotic cell cycle, a combination of biophotonic analyses with RNA interference was employed. Immunofluorescence reveals that TIP150 localizes to the outer most part of the kinetochore and to the ends of growing microtubules by its C-terminal domain. Suppression of TIP150 resulted in an increase in mitotic defects, reduced kinetochore-microtubule attachment, and perturbed chromosome oscillation dynamics. Furthermore, the use of total internal reflection microscopy reveals a novel role for TIP150-EB1 elicited tracking to the microtubule ends. We reason that TIP150 is essential for kinetochore-microtubule attachment in mitosis and microtubule dynamics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2056. doi:1538-7445.AM2012-2056