We evaluated the protective effect of a tocotrienol-rich fraction (TRF) from grape seeds on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. Generation of cellular reactive oxygen species (ROS), concentrations of cellular lipid peroxidation products and reduced glutathione, and antioxidant enzyme activity were used as biomarkers of cellular oxidative status. Cells pretreated with TRF (10–500 μg/mL) showed an increased resistance to oxidative stress in a dose-dependent manner, as revealed by a higher percentage of surviving cells compared to control cells. Pretreatment with TRF (5–100 μg/mL) prevented the decrease in reduced glutathione and the increase in malondialdehyde and ROS evoked by TBHP in HepG2 cells. Moreover, TRF pretreatment prevented a significant increase in glutathione peroxidase, catalase, and superoxide dismutase activities induced by TBHP. These results show that TRF has significant protective ability against TBHP-induced oxidative insult and that the modulation of antioxidant enzymes by TRF may have an important antioxidant effect on HepG2 cells.