Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either p53 or PUMA led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome c release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor–induced apoptosis in colon cancer cells. [Mol Cancer Ther 2007;6(3):1062–9]