This study was conducted to investigate the effect of Nelumbinis Plumula total alkaloid (NPA) and its main alkaloid components on oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in the human hepatocellular HepG2 cell line. According to HPLC analysis, several major alkaloid compounds such as liensinine, isoliensinine and neferine were present in NPA. The cytotoxic effects in 0.55 mM t-BHP-induced HepG2 cells were significantly inhibited by NPA and the major compound in NPA, neferine, showed the strongest activities. The protective effect of neferine against oxidative stress induced by t-BHP may be associated with decreased ROS formation, TBARS generation, LDH release and increased GSH levels, suggesting their involvement of the cytoprotective on oxidative stress. The effects were comparable with quercetin, which was used as positive control. Overall, total alkaloid and alkaloid compounds from Nelumbinis Plumula displayed a significant cytoprotective effect against oxidative stress. Further study is needed to elucidate the relationship between the chemical structures of the components in NPA and their protective effect on oxidative stress.