Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor with poor survival rate. Temozolomide (TMZ) is used as standard chemotherapy to treat GBM, but a large number of patients either respond poorly and/or develop resistance after long-term use, emphasizing the need to develop potent drugs with novel mechanisms of action. Here, using high-throughput compound screening (HTS), we found that azathioprine, an immunosuppressant, is a promising therapeutic agent to treat TMZ-resistant GBM. Through integrative genome-wide analysis and global proteomic analysis, we found that elevated lipid metabolism likely due to hyperactive EGFR/AKT/SREBP-1 signaling was inhibited by azathioprine. Azathioprine also promoted ER stress-induced apoptosis. Analysis of orthotopic xenograft models injected with patient-derived GBM cells revealed reduced tumor volume and increased apoptosis after azathioprine and TMZ co-treatment. These data indicate that azathioprine could be a powerful therapeutic option for TMZ-resistant GBM patients.