Glioblastoma is one of the most angiogenic human tumors and characterized by microvascular proliferations. A better understanding of glioblastoma vasculature is needed to optimize anti-angiogenic therapy that has shown a promising but incomplete efficacy. The present study examined 48 glioblastomas by CD34 endothelial marker periodic acid-Schiff (PAS) dual staining and found non-endothelial cell-lined blood vessels that were formed by tumor cells (vasculogenic mimicry, VM) existing in a fraction of these tumors. We hypothesized that CD133-positive glioblastoma stem-like cells (GSCs) may play a pivotal role in glioblastoma VM formation and then demonstrated in vitro and in vivo that a subset of GSCs were capable of vasculogenesis. Moreover, we found that several growth factors involved in normal angiogenesis were expressed in GSCs. We describe here a new mechanism of alternative glioblastoma vascularization and open a new perspective for the anti-vascular treatment strategy.