Glioblastoma multiforme (GBM) is a life-threatening brain tumor with fatal recurrence, for which glioblastoma stem cells (GSCs) are held responsible. Though endothelial-monocyte activating polypeptide-II (EMAP-II) has been confirmed as a possible antitumor agent that can induce apoptosis of endothelial cells and inhibit tumor angiogenesis, the direct cytotoxicity by EMAP-II on tumor cells and its underlying mechanism are largely unknown. In the present study, it was demonstrated that low-dose (0.05 nM) EMAP-II reduces cell viability and mitochondrial membrane potential in vitro. Likewise, EMAP-II suppressed tumor growth in GSC-xenografted mice. Though no apoptosis was detected, all these antitumor effects were attenuated when GSCs were pretreated with 3-methyladenine (3-MA). Analysis of EMAP-II-treated GSCs exhibited the morphological and biochemical changes typical of autophagy, which was further shown to be defective. Moreover, EMAP-II was found to suppress tumor growth by inducing G2/M arrest in GSCs. Our data further showed that EMAP-II inhibited PI3K/Akt activation with concomitant induction of FoxO1 activation. FoxO1 knockdown significantly attenuated the induction of autophagy and G2/M arrest. Excessive accumulation of lipid droplets was intriguingly detected by transmission electron microscope, which was accompanied by autophagosomes. Further investigation indicated that the transcriptional regulation of Atg2B by FoxO1 was responsible for the induction of autophagy and formation of lipid droplets. These results suggest that EMAP-II is an effective anticancer agent for glioblastoma therapy, which can induce direct growth suppression in GSCs through defective autophagy and G2/M arrest mediated by the PI3K/Akt/FoxO1 axis.