Astrocyte elevated gene-1 (AEG-1) has been explored as a novel target for human glioma therapy, thus reflecting its potential contribution to gliomagenesis. In the present study, we investigated the effect of DYT-40, a novel synthetic 2-styryl-5-nitroimidazole derivative, on cell growth and invasion in glioblastoma (GBM) and uncovered the underlying mechanisms of this molecule. DYT-40 induces the intrinsic mitochondrial pathway of apoptosis and inhibits the epithelial-mesenchymal transition (EMT) and invasion of GBM cell lines. Furthermore, DYT-40 deactivates PI3K/Akt and MAPK pathways, suppresses AEG-1 expression, and inhibits NF-κB nuclear translocation. DYT-40 reduced the tumor volumes in a rat C6 glioma model by apoptotic induction. Moreover, HE staining demonstrated that the glioma rat model treated with DYT-40 exhibited better defined tumor margins and fewer invasive cells to the contralateral striatum compared with the vehicle control and temozolomide-treated rats. Microscopic examination showed a decrease in AEG-1-positive cells in DYT-40-treated rats compared with the untreated controls. DYT-40-treatment increases the in vivo apoptotic response of glioma cells to DYT-40 treatment by TUNEL staining. In conclusion, the inhibitory effects of DYT-40 on growth and invasion in GBM suggest that DYT-40 might be a potential AEG-1 inhibitor to prevent the growth and motility of malignant glioma.