We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF(2α)), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF(2α) and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.