Extracellularly added recombinant granulysin was reported to kill mammalian target cells. The sites of actions and molecular mechanisms of granulysin in target cell killing, however, are presently unclear. In order to provide new insights into its potential mechanism of target cell damage, we here constructed recombinant plasmids carrying 9 kDa granulysin cDNA and examined effects of intracellularly expressed granulysin on the target hepatoma SMMC-7721 cells. The localization of intracellularly expressed granulysin was examined by fluorescence microscopy and confocal microscopy. Effects of granulysin on cell proliferation and apoptosis were measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, flow cytometry and fluorescence microscopy, respectively. Changes of mitochondrial membrane potential were monitored by fluorescence microscopy. On the other hand, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF) was evaluated by Western blot and confocal microscopy. Intracellularly expressed granulysin was preferentially localized in cytoplasm, noticeably inhibited cell proliferation and induced cell death accompanied by reduced mitochondrial membrane potential, release of AIF and cytochrome c from mitochondria. Taken together, our findings demonstrate for the first time that localization and effect of intracellularly expressed granulysin on non-native cancer cells and indicate its potential utility in gene therapy for cancer.