The antioxidative fraction of roasted pearl barley (adlay, Coix lacryma-jobi L. var Ma-yuen Stapf), which is used in commercial tea beverages, was obtained by column chromatography and eluted with 70% ethanol. We investigated the effect of dietary supplementation with pearl barley extract on the formation of thiobarbituric acid-reactive substances in rat serum and ex vivo tissues. We also investigated whether the serum and tissue homogenates were susceptible to lipid peroxidation. Rats that were fed a diet containing 5 g/kg pearl barley extract (antioxidative fraction) for 14 days exhibited no significant reduction in the formation of thiobarbituric acid-reactive substances in the serum. However, compared to controls, liver homogenates from rats fed pearl barley extract showed significantly lower susceptibility to lipid peroxidation (induced by 2,2′-azobis(2-amidinopropane)hydrochloride). These results demonstrate that a diet containing pearl barley extract increases oxidation resistance in ex vivo rat livers.