Polyphyllin VI, which is an active saponin, is mainly isolated from traditional medicinal plant Paris polyphylla, which causes liver damage in rats. In the present study, we aimed to explore the potential cytotoxicity of polyphyllin VI on the growth of HepaRG cells and to determine the molecular mechanism. The results revealed that polyphyllin VI changed cell morphology and induced apoptosis in HepaRG cells. Flow cytometric assay displayed that polyphyllin VI promoted the generation of reactive oxygen species (ROS), depolarized the mitochondrial membrane potential (MMP), and induced S phase cell cycle arrest by decreasing the expression of cyclin A2 and CDK2, while significantly increasing the expression of p21 protein. Polyphyllin VI induced the release of cytochrome c from the mitochondria to the cytosol and activated Fas, caspase-3, -8, -9, and PARP proteins. Pretreatment with NAC and Z-VAD-FMK (ROS scavenger and caspase inhibitor, respectively) on HepaRG cells increased the percentage of viable cells, which indicated that polyphyllin VI induced cell apoptosis through mitochondrial pathway by the generation of ROS and Fas death-dependent pathway. All of the effects are in dose- and time-dependent manners. Taken together, these findings emphasize the necessity of risk assessment to polyphyllin VI and offer an insight into polyphyllin VI-induced apoptosis of HepaRG cells.