Abstract Purpose: Searching for novel strategies to modulate apoptosis in neuroblastoma, we investigated the potential of the proteasome inhibitor bortezomib. Experimental Design: The effect of bortezomib on TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis signaling pathways was analyzed in neuroblastoma cell lines, primary neuroblastoma cultures, and in an in vivo model. Results: Bortezomib synergistically cooperates with TRAIL to induce apoptosis and to reduce colony formation of neuroblastoma cells (combination index: 0.5). Mechanistic studies reveal that bortezomib profoundly enhances TRAIL-induced cleavage of Bid into tBid, accumulation of tBid in the cytosol, and its insertion into mitochondrial membranes, pointing to a concerted effect on Bid cleavage (TRAIL) and stabilization of tBid (bortezomib), which links the death receptor to the mitochondrial pathway. In addition, bortezomib increases expression of p53 and Noxa. All these changes lead to increased activation of Bax and Bak, loss of the mitochondrial membrane potential, cytochrome c release, caspase activation, and caspase-dependent apoptosis on treatment with bortezomib and TRAIL. Knockdown of Bid, Noxa, or p53 significantly delays the kinetic of bortezomib- and TRAIL-induced apoptosis, whereas it does not confer long-term protection. By comparison, overexpression of Bcl-2, which simultaneously antagonizes tBid and p53, significantly inhibits bortezomib- and TRAIL-induced apoptosis and even rescues clonogenic survival. Importantly, bortezomib and TRAIL act in concert to trigger apoptosis and to suppress tumor growth in patient-derived primary neuroblastoma cells and in an in vivo model of neuroblastoma. Conclusions: Bortezomib represents a promising new approach to prime neuroblastoma cells toward TRAIL, which warrants further investigation. Clin Cancer Res; 17(10); 3204–18. ©2011 AACR.