Glioblastoma (GB) remains a devastating disease for which novel therapies are urgently needed. Here we report that the Aurora-A kinase inhibitor alisertib exhibits potent efficacy against GB neurosphere tumor stem-like cells in vitro and in vivo. Many GB neurosphere cells treated with alisertib for short periods undergo apoptosis, although, some regain proliferative activity upon drug removal. Extended treatment however results in complete and irreversible loss of tumor cell proliferation. Moreover, alisertib caused GB neurosphere cells to partially differentiate and enter senescence. These effects were also observed in glioma cells treated with the Aurora-A inhibitor TC-A2317 or anti-Aurora-A siRNA. Furthermore, alisertib extended median survival of mice bearing intracranial human GB neurosphere tumor xenografts. Alisertib exerted similar effects on GB neurosphere cells in vivo based on the presence of activated phosphoThr288Aurora-A, abnormal mitoses and increased cellular ploidy, consistent with on-target activity. Our results offer preclinical proof-of-concept for alisertib as a new therapeutic for glioma treatment.