Mitotic spindle dynamics are highly dependent on proteins that interact with microtubules to influence their organization or stability. Here, we show that the Drosophila Toucan protein interacts directly with microtubules. Its localization to the microtubule network when it is expressed in mammalian cells and its direct interaction with microtubules in vitro are dependent on its central basic domain. Moreover, Toc expression in mammalian cells strongly protects microtubules from depolymerization. By using in vivo inducible RNAi in syncytial embryos, we generated a dose-sensitive loss of function of toucan, demonstrating that this technique is an efficient method for inactivating a maternal transcript. This enabled us to accurately characterize several new mitotic defects from the early to the late phases of mitosis, depending on Toucan depletion level. Toucan is required for metaphase spindle formation and centrosome anchoring to the poles. Then, during anaphase, Toc depletion affects kinetochore microtubules and therefore chromosome segregation. Toc is also necessary for central spindle formation by the interpolar microtubules. In contrast, astral microtubules are not disturbed by Toc depletion. Taken together, our results show that Toucan is a microtubule-associated protein specifically required for the stability of spindle microtubules throughout mitosis.