Glioblastoma multiforme (GBM) is the most common type of malignant brain tumor. The present standard treatment for GBM has not been effective; therefore, the prognosis remains dramatically poor and prolonged survival after treatment is still limited. The new therapeutic strategies are urgently needed to improve the treatment efficiency. Doxorubicin (Dox) has been widely used in the treatment of many cancers for decades. In recent years, with the advancement of delivery technology, more and more research indicates that Dox has the opportunity to be used in the treatment of GBM. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), has been reported to have oncogenic effects in many cancer cell types and is implicated in drug resistance. However, the biological function and molecular mechanism of AREG in Dox treatment of GBM are still unclear. Here, we demonstrate that knockdown of AREG can boost Dox-induced endoplasmic reticulum (ER) stress to trigger activation in both autophagy and apoptosis in GBM cells, ultimately leading to cell death. To explore the importance of AREG in the clinic, we used available bioinformatics tools and found AREG is highly expressed in GBM tumor tissues that are associated with poor survival. In addition, we also used antibody array analysis to dissect pathways that are likely to be activated by AREG. Taken together, our results revealed AREG can serve as a potential therapeutic target and a promising biomarker in GBM.