Glioblastoma (GBM) is the most aggressive and lethal type of tumor in the central nervous system, characterized by a high incidence and poor prognosis. Thiotert, as a novel dual targeting agent, has potential inhibitory effects on various tumors. Here, we found that Thiotert effectively inhibited the proliferation of GBM cells by inducing G2/M cell cycle arrest and suppressed the migratory ability in vitro. Furthermore, Thiotert disrupted the thioredoxin (Trx) system while causing cellular DNA damage, which in turn caused endoplasmic reticulum (ER) stress-dependent autophagy. Knockdown of ER stress-related protein ATF4 in U251 cells inhibited ER stress-dependent autophagy caused by Thiotert to some extent. Orthotopic transplantation experiments further showed that Thiotert had the same anti-GBM activity and mechanism as in vitro. Conclusively, these results suggest that Thiotert induces ER stress-dependent autophagy in GBM cells by disrupting redox homeostasis and causing DNA damage, which provides new insight for the treatment of GBM.