Temozolomide is universally used to treat glioblastoma due to its unique ability to cross the blood-brain barrier and inhibit tumor growth through DNA alkylation. However, over time, the inevitable emergence of resistance to temozolomide impedes successful treatment of this cancer. As a result, there is an urgent need to identify new therapeutic targets to improve treatment outcomes for this malignancy. In this work, acquired temozolomide-resistant glioblastoma cell lines LN18 (LN18-TR) and T98G (T98G-TR) exhibited stronger aggressiveness and lower endoplasmic reticulum (ER) stress than their parental cells.. Besides, temozolomide resistance was associated with elevated proteasome activity that suppressed ER stress, which was restored upon inhibition of the proteasome with MG132. Specifically, our study revealed that the 19S proteasomal regulatory subunit PSMC2, which was overexpressed in adapted temozolomide-resistant glioblastoma cells, reduced pro-death autophagy and decreased temozolomide sensitivity in parental cells when overexpressed. While autophagy increased in parental cells following temozolomide treatment, it was not elevated in temozolomide-resistant glioblastoma cells. Genetic suppression of PSMC2 triggered the JNK signalling pathway causing phosphorylation of BCL2, allowing Beclin1 to be released from the BCL2-Beclin1 complex. This boosted autophagosome nucleation, increased pro-death autophagy, and restored apoptosis in temozolomide-resistant glioblastoma cells. Finally, targeting PSMC2 provided a unique method for interrupting autophagy-mediated ER stress maintenance and temozolomide resistance in glioblastoma.