Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins. The proteasome inhibitor bortezomib (PS-341, Velcade) induces apoptosis in various malignant cells and has been approved for treatment of refractory multiple myeloma. Inhibition of the antiapoptotic transcription factor nuclear factor-κB (NF-κB) apparently contributes to the antitumor effects of bortezomib; however, this mechanism cannot fully explain the exceptional sensitivity of myeloma cells. Extensive protein synthesis as in myeloma cells is inherently accompanied by unfolded proteins, including defective ribosomal products (DRiPs), which need to be degraded by the ubiquitin-proteasome system. Therefore, we hypothesized that the proapoptotic effect of bortezomib in multiple myeloma is mainly due to the accumulation of unfolded proteins in cells with high protein biosynthesis. Using the IgG-secreting human myeloma cell line JK-6L and murine μH-chain–transfected Ag8.H myeloma cells, apoptosis induction upon proteasome inhibition was clearly correlated with the amount of immunoglobulin production. Preferentially in immunoglobulin-high myeloma cells, bortezomib triggered activation of caspases and induction of proapoptotic CHOP, a component of the terminal unfolded protein response induced by endoplasmic reticulum (ER) stress. In immunoglobulin-high cells, bortezomib increased the levels of proapoptotic Bax while reducing antiapoptotic Bcl-2. Finally, IgG-DRiPs were detected in proteasome inhibitor–treated cells. Hence, proteasome inhibitors induce apoptosis preferentially in cells with high synthesis rate of immunoglobulin associated with accumulation of unfolded proteins/DRiPs inducing ER stress. These findings further elucidate the antitumor activities of proteasome inhibitors and have important implications for optimizing clinical applications. [Cancer Res 2007;67(4):1783–92]