Diallyl trisulfide (DATS) is known to have an anticancer effect on human cancer cells. However, the exact mechanisms of this anticancer activity remain unclear. To explore the effect of DATS on human hepatocellular carcinoma HepG2 cell apoptosis. HepG2 cells were either treated with 50 μmol/L, 100 μmol/L of DATS for 0, 6, 12，24，48 and 72 h, respectively. The mitochondrial membrane potential was visualized by the JC-1 fluorescence staining. The cytochrome c in HepG2 cells was detected by Western blotting. The activity of caspase-3 in HepG2 cells was measured with ELISA. Diallyl trisulfide induces HepG2 cell apoptosis. The apoptosis percentage in HepG2 cells is up to 60.33% and 93.67%, respectively, when the cell was treated with 50 μmol/L or 100 μmol/L of DATS for 48 h. Diallyl trisulfide degrades the mitochondrial membrane potential and lowers the cytochrome c in mitochondria while increases the cytochrome c in the cytoplasma that mitochondria was deleted. Diallyl trisulfide activates caspase-3 kinase in HepG2 cells. These results indicate that diallyl trisulfide induces apoptosis by decreasing mitochondrial membrane potential, facilitating cytochrome c release from mitochondria into cytoplasm, and activating caspase-3 kinase in human hepatocellular carcinoma HepG2 cell.