Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-anaphase-promoting complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma. Mao et al. report that E3 ubiquitin ligase CDC20-APC is required for invasiveness, self-renewal, and in vivo tumorigenicity of human glioblastoma stem-like cells (GSCs). CDC20-APC interacts with and regulates SOX2 protein to promote SOX2-dependent transcription and drive GSC invasiveness and self-renewal. Using the Cancer Genome Atlas dataset, the authors find that high CDC20 expression in proneural glioblastomas is associated with shorter overall survival.