Glioblastoma is refractory to current treatment approaches calling for novel strategies. One emerging option is proteasome inhibition as some antitumor activity in glioblastoma has recently been shown. Here, we report that the proteasome inhibitor Bortezomib primes glioblastoma cells including glioblastoma stem cells to TRAIL-induced apoptosis in vitro and in vivo by increasing tBid stability and facilitating mitochondrial apoptosis. Investigations into the underlying molecular mechanisms reveal that Bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid, and the stability of TRAILderived tBid markedly increases upon proteasome inhibition. Notably, knockdown of Bid significantly decreases Bortezomib- and TRAIL-mediated apoptosis. By comparison, silencing of Noxa, which is also upregulated by Bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in co-treated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and apoptosis, underscoring the functional relevance of the mitochondrial pathway for apoptosis induction. In addition, Bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, demonstrating an effect on long-term survival. Of note, Bortezomib profoundly enhances TRAIL-triggered apoptosis in primary cultured glioblastoma cells and in patients-derived glioblastoma stem cells, underlining the clinical relevance of this combination. Importantly, Bortezomib cooperates with TRAIL to suppress glioblastoma growth in an in vivo model of glioblastoma. In conclusion, these findings provide compelling evidence that the combination of Bortezomib and TRAIL presents a promising novel strategy to trigger apoptosis in glioblastoma including glioblastoma stem cells, which warrants further investigation.