Despite their feasibility as food flavouring, glycidol is classified as a probable carcinogen under group 2A by WHO. The cytotoxicity effects of isomers of R- and S-glycidol on African green monkey kidney normal cell lines (Vero) and human colon cancer cell line (HCT 116) remain unclear. Cell viability of the treated Vero and HCT 116 cells was determined using the AlamarBlue® assay. Dichlorodihydrofluorescein diacetate (DCFDA) was used to evaluate reactive oxygen species (ROS) activity. Protein expressions of ERK ½, p-ERK, Bcl-2 and caspase-3 were investigated using western blotting technique. The findings indicated that R- and S-glycidol (1.16 μg/mL) exposure dramatically reduced the cell viability of the treated HCT 116 cells but was slightly cytotoxic to Vero cells, hence triggering ROS activity. R- and S-glycidol cause down-regulation of ERK ½, p-ERK, and BCL-2 protein expression at 48 h of treatment. Furthermore, both R- and S-glycidol possess close interaction in proximity to 3D-structure of human ERK and p-ERK protein receptors. In conclusion, R- and S-glycidol potentially triggered oxidative stress and affected ERK protein phosphorylation, leading to caspase-3 independent cell death of the treated HCT 166 cells, suggesting that lower doses (<1.16 μg/mL) of R- and S-glycidol are safe for human consumption.