The mitotic spindle is a microtubule-based apparatus that is responsible for accurate segregation of chromosomes into two daughter cells. In this study, we show that the DNA damage kinase Chk1 is required for optimal density and efficient polymerization of spindle microtubules during unperturbed mitosis in vertebrate cells. Chk1 phosphorylates -tubulin at the newly identified site threonine-285 (T285) at mitotic centrosomes. Impaired -tubulin-T285 phosphorylation correlates with improper spindles, delayed anaphase onset, erroneous chromosome alignment and segregation, unequal daughter cell-size and reduced cell proliferation, whereas expression of a phosphomimetic -tubulin rescues spindle formation and functions in Chk1-deficient cells. The ATR-interacting protein ATRIP promotes localization of the ATR kinase and mediator protein TopBP1 to mitotic centrosomes; furthermore, interaction of ATRIP with ATR and TopBP1 is required for Chk1 activation and - tubulin-T285 phosphorylation. Also, inhibition of T285 phosphorylation synergizes with microtubule poisons to inhibit cell proliferation. These results identify a novel mechanism that promotes normal spindle formation, through Chk1-mediated - tubulin-T285 phosphorylation.