ABSTRACT The objective of this experiment was to investigate the effect of Aspergillus oryzae α-amylase (AAM) supplementation on rumen VFA profile and relative abundance of mRNA associated with nutrient absorption in ruminal and duodenal tissue from beef steers. Nine crossbred beef steers (BW 622 ± 50 kg), fitted with rumen and duodenal fistulas, were used in this experiment. Steers were housed in individual stanchions and fed a high-concentrate finishing diet (6% forage NDF) twice daily for 8 d. Treatments included (1) control (corn meal; n = 5) and (2) AAM (fungal α-amylase 750 units/g; n = 4). Dietary treatment supplements were manufactured before each feeding by mixing 3 g of α-amylase or corn meal into 150 g of dried distillers grains for the morning feeding and 2 g of α-amylase or corn meal into 100 g of dried distillers grains for the afternoon feeding. Supplements were applied as a top dress for every feeding and thoroughly mixed by hand. On d 5, rumen fluid samples were obtained every 4 h for 24 h and analyzed for VFA. On d 9, rumen papillae and duodenal mucosal tissue samples were collected. Total tissue RNA was extracted for real-time PCR analysis. Sodium/potassium ATPase pump α1; glucose transporter 2 and 5; putative anion transporter isoform 1; sodium/hydrogen antiporter isoforms 1, 2, and 3; 3-hydroxy 3-methylglutaryl coenzyme A synthase isoform 2; downregulated in adenoma; monocarboxylate cotransporter isoform 1; and glyceraldehyde-3-phosphate dehydrogenase mRNA were tested. No treatment differences (P > 0.21) were detected for any of the genes analyzed in ruminal or duodenal tissue. Concentrations of VFA and the acetate-to-propionate ratio did not differ (P > 0.22) among treatments. However, the acetate-to-propionate ratio and molar percentages of butyrate were numerically greater (P = 0.17) in AAM steers compared with controls. Under the conditions of this experiment, AAM supplementation had no effect on relative expression of mRNA associated with nutrient absorption and minimal effects on molar proportions of VFA.