Hepatic stimulator substance (HSS) has been shown to protect liver cells from various toxins. However, the mechanism by which HSS protects hepatocytes remains unclear. In this study, we established BEL-7402 cells that stably express HSS and analyzed the protective ability of HSS on cells through mitochondrial permeability (MP). After administration of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a specific agent that leads to depolarization of the mitochondrial transmembrane potential, the apoptosis rate of HSS-expressing cells was significantly reduced, as measured using Hoechst staining and flow cytometry. The mitochondrial membrane transition and cytochrome c leakage were significantly inhibited in the HSS-expressing cells as compared with the untransfected cells, and, as a consequence, the cellular ATP content in the HSS-expressing cells was relatively preserved. Additionally, decreased caspase-3 activity was observed in the HSS-expressing cells treated with CCCP as compared with the vector-transfected cells and cells expressing mutant HSS. Furthermore, silencing of HSS expression using small interfering RNA accelerated CCCP-induced apoptosis. In isolated mitochondria, recombinant HSS reduced the release of cytochrome c induced by CCCP, indicating a possible role for HSS in regulation of mitochondrial permeability transition (MPT). HSS-expressing BEL-7402 cells are resistant to CCCP injury, and HSS protection is identical to that observed with cyclosporin A, an inhibitor of MPT. Therefore, we propose that the protective effect of HSS may be associated with blockade of MPT.