The proteasome-mediated protein degradation is critical for regulation of a variety of cellular processes, including cell cycle, cell death, differentiation and immune response. Proteasome inhibitors have recently been shown to be potent anti-cancer agents against a variety of cancer cells. Our study demonstrated that proteasome inhibitor MG132 (carbobenzoxy-L-leucyle-L-leucyl-L-leucinal) was a potent death-inducing agent for PC3 prostate cancer cells. MG132-induced cell death was partially inhibited by pan-caspase inhibitor zAVD-fmk and translational inhibitor cycloheximide. To understand the signaling pathways of proteasome inhibitor-induced cell death, we performed gene profiling study using Affymetrix human DNA microarrays to identify the genes whose expression was affected by proteasome inhibitor MG132 in PC3 cells. The genes with more than threefold increased expression induced by MG132 were functionally categorized into the following groups: heat shock and chaperone proteins, ubiquitination and protein degradation, transcription/translation factors, cell death and cell cycle arrest, signaling molecules and enzymes, and secreted cytokines. Among them, heat shock proteins and anti-oxidant enzymes may promote cell survival, while pro-death proteins such as GADD45B and STK17a may promote cell death. Interestingly, expression of a few autophagic genes was elevated by MG132 treatment. Furthermore, autophagy inhibitor 3-methyladenine partially inhibited MG132-induced cell death, indicating that autophagic cell death may contribute to MG132-induced cell death. Taken together, our results demonstrated that proteasome inhibition elicits activation of multiple signaling pathways in prostate cancer cells.