The current study was designed to investigate possible protective effect of allyl isothiocyanate (AITC) in nitrite- and nitrosamine-treated human hepatoma cells (HepG2) with the evaluation by cytotoxic effects and genotoxic effects determined by the single-cell gel electrophoresis (SCGE). Allyl isothiocyanate treatment enhanced cell viability and reduced intracellular reactive oxygen species (ROS) production in both nitrite- and nitrosamine-treated cells significantly. In SCGE, when compared to untreated control cells, all of the treated groups caused increases in the tail intensity (%) such as nitrite at 17%, N-nitrosodimethylamine (NDMA) at 279%, N-nitrosodiethylamine (NDEA) at 324%, and N-nitrosomorpholine (NMOR) at 288%. Allyl isothiocyanate reduced the tail intensity caused by nitrite 36%, by NDMA 36%, by NDEA 49%, and by NMOR 32%, respectively, when compared to each individual toxic compound-treated group. In conclusion, AITC protected HepG2 cells against cytotoxic and genotoxic effects caused by nitrite and the nitrosamines.