Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop and is reported to protect against various diseases such as cancer. To investigate the effect of the ethanolic extract of adlay bran (ABE) on drug-metabolizing enzymes and glutathione-related antioxidant enzymes in rats, three groups of eight male Sprague-Dawley rats each were fed a control diet or a diet containing 5 or 10% ABE for 4 weeks. Significant decreases in microsomal cytochrome P-450 (CYP) 1A1-catalyzed ethoxyresorufin O-deethylation, CYP2C-catalyzed diclofenac 4-hydroxylation, CYP2D-catalyzed dextromethorphan O-demethylation, and CYP3A-catalyzed testosterone 6β-hydroxylation in the liver and CYP1A1-catalyzed ethoxyresorufin O-deethylation in the lungs of rats fed ABE were observed. Immunoblot analyses also showed decreases of CYP1A1, 1A2, 2C6, 2C11, 2D1, 2E1, 3A1, and 3A2 in the liver and CYP1A1 in the lungs. Furthermore, rats fed the 10% ABE diet had a higher glutathione content and glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities in the lungs, but such an increase was not noted in the liver. Inhibition of various CYP-catalyzed enzyme reactions by ABE in rat and human liver microsomes had also been shown. The results of this study indicate that ABE feeding may suppress CYP enzyme activities and CYP protein expression in the liver and lungs of rats. Moreover, the increase of the antioxidant potential by ABE is tissue-specific.