The antioxidant potential of carotenoids from aleurone, germ, and endosperm fractions of barley, corn, and wheat has been evaluated. HPLC analysis confirmed the presence of lutein and zeaxanthin carotenoids (nd-15139 μg/kg) in extracts of cereal grain fractions. The antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorbance capacity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays revealed significantly higher (P<0.001) antioxidant activity in the germ than in the aleurone and endosperm fractions. Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,2'azobis (2-amidinopropane)dihydrochloride (AAPH)-induced cell loss was effectively reduced by preincubating Caco-2, HT-29, and FHs 74 Int cells with carotenoid extracts. Moreover, carotenoid extracts reduced (P<0.001) AAPH-induced intracellular oxidation in the cell lines, suggesting antioxidant activity. Of the 84 antioxidant pathway genes included in microarray array analysis (HT-29 cells), the expressions of 28 genes were enhanced (P<0.05). Our findings suggest that carotenoids of germ, aleurone, and endosperm fractions improved antioxidant capacity and thus have the potential to mitigate oxidative stress.