Bortezomib (PS-341), a selective inhibitor of proteasomes, induces apoptosis in multiple myeloma (MM) cells; however, prolonged drug exposure may result in cumulative toxicity and the development of chemoresistance. Here we show that combining PK-11195 (PK), an antagonist to mitochondrial peripheral benzodiazepine receptors (PBRs), with bortezomib triggers synergistic anti-MM activity even in doxorubicin-, melphalan-, thalidomide-, dexamethasone-, and bortezomib-resistant MM cells. No significant cytotoxicity was noted in normal lymphocytes. Low-dose combined PK and bortezomib treatment overcomes the growth, survival, and drug resistance conferred by interleukin-6 or insulin growth factor within the MM bone marrow milieu. The mechanism of PK + bortezomib-induced apoptosis includes: loss of mitochondrial membrane potential; superoxide generation; release of mitochondrial proteins cytochrome-c (cyto-c) and Smac; and activation of caspases-8/-9/-3. Furthermore, PK + bortezomib activates c-Jun NH2 terminal kinase (JNK), which translocates to mitochondria, thereby facilitating release of cyto-c and Smac from mitochondria to cytosol. Blocking JNK, by either dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor SP600125, abrogates both PK + bortezomib-induced release of cyto-c/Smac and induction of apoptosis. Together, these preclinical studies suggest that combining bortezomib with PK may enhance its clinical efficacy, reduce attendant toxicity, and overcome conventional and bortezomib resistance in patients with relapsed refractory MM.