Inhibitors of the proteasome, a multicatalytic proteinase complex responsible for intracellular proteolysis, activate programmed cell death in part through the c-Jun-N-terminal kinase (JNK). Proteasome inhibitors also induce mitogen-activated protein kinase phosphatase-1 (MKP-1), however, which can inactivate JNK, and we therefore considered the hypothesis that MKP-1 induction may be antiapoptotic. Over-expression of MKP-1 in A1N4-myc human mammary epithelial and BT-474 breast carcinoma cells decreased proteasome inhibitor-mediated apoptosis. On the other hand, BT-474 cells stably expressing an MKP-1 small interfering RNA (siMKP-1) and MKP-1 knockout mouse embryo fibroblasts underwent enhanced apoptosis compared with their respective controls. MKP-1-mediated inhibition of apoptosis was associated with decreased phospho-JNK levels, whereas MKP-1 suppression or inactivation enhanced phospho-JNK. Anthracyclines repress MKP-1 transcription, suggesting that they could enhance proteasome inhibitor-mediated apoptosis. Such combinations induced increased cell death in association with enhanced phospho-JNK and decreased MKP-1 levels. Inhibition of JNK signaling decreased the proapoptotic activity of the anthracycline/proteasome inhibitor regimen. Xenograft studies showed the combination was more effective at inducing tumor growth delay, associated with suppression of MKP-1 and enhancement of apoptosis and phospho-JNK. Infection of anthracycline/proteasome inhibitor-treated A1N4-myc cells with Adenoviral-MKP-1 suppressed apoptosis and phospho-JNK. Finally, the anthracycline/proteasome inhibitor regimen activated apoptosis and phospho-JNK to a greater extent in BT-474/siMKP-1 cells than controls. These findings for the first time demonstrate that proteasome inhibitor-mediated induction of MKP-1 is antiapoptotic through inhibition of JNK. Furthermore, they suggest that a proteasome inhibitor/anthracycline regimen holds potential for enhanced antitumor activity in part through repression of MKP-1, supporting clinical evaluation of such combinations.