Addition of proteasome inhibitor PS-341 (VELCADE, bortezomib) to prostate cancer cells enhances cell death mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). PS-341 sensitizes prostate cancer cells to TRAIL-induced apoptosis by increasing TRAIL receptors (DR5), inhibiting protein degradation, and elevating DR5 mRNA. Investigations into how PS-341 regulates the stability of DR5 mRNA revealed that PS-341 increased DR5 mRNA by extending its half-life from 4 to 10 h. The 2.5-kb 3'-untranslated region of the DR5 gene stabilized a heterologous gene in LNCaP human prostate cancer cells, suggesting the importance of this mRNA sequence. In contrast, human prostate cancer cell lines PC-3 and DU145 do not show this stabilization, suggesting cell specificity. PS-341 treatment of LNCaP cells increases the level of specific cytoplasmic mRNA-binding proteins, including AUF-1 isoforms, hnRNP C1/C2, and HuR proteins. In UV cross-linking experiments, after PS-341 treatment, the HuR protein markedly increases binding to specific sequences in the DR5 3'-untranslated region. In LNCaP cells treated with PS-341, small interfering RNA-mediated knockdown of HuR markedly decreases the half-life of DR5 mRNA, indicating that HuR is essential for mRNA stabilization. HuR protein is ubiquitinated, suggesting that PS-341 increases this protein by preventing its degradation. These experiments implicate modulation of mRNA stability as a novel mechanism by which proteasome inhibitors function, sensitizing cancer cells to antineoplastic agents.