Abstract We examined the effects of dietary supplementation of a Saccharomyces cerevisiae-based direct-fed microbial on plasma carbonyl-metabolome and fecal bacterial community of beef steers during a 42-d receiving period. Forty newly-weaned beef steers were used in this study. The steers were stratified by BW and randomly assigned to receive a basal diet with no additive (CON; n = 20) or a basal diet supplemented with 19 g of CommenceTM (PROB; n = 20). CommenceTM (PMI, Arden Hills, MN) contains a blend of 6.2 × 1011 cfu/g of S. cerevisiae, 3.5 × 1010 cfu/g of a mixture of Enterococcus lactis, Bacillus subtilis, Enterococcus faecium, and L. casei. On d 0 and 40, rectal fecal samples were collected for bacterial community analysis via sequencing of the V3-V4 region of 16S rRNA gene. On d 42, blood was collected for analysis of carbonyl-containing metabolites in plasma using a chemical isotope labeling/liquid chromatography mass spectrometry-based untargeted metabolomics. The data were analyzed using the GLIMMIX procedure of SAS with treatment as a fixed effect) and (random effect). A total number of 812 unique plasma metabolites were detected. Up to 305 metabolites [fold change (FC) ≥ 1.5, FDR ≤ 0.01] including glucose, hippuric acid, glycoaldehyde, and 5-hydroxykynurenamine were increased by PROB supplementation, whereas 199 metabolites (FC ≤ 0.63, FDR ≤ 0.01) including acetoacetate were reduced. Correlation analysis showed that plasma concentrations of 5-oxopentanoate, 2-aceto-2-hydroxybutanoate, and 3-methyl-2-oxopentanoic acid were positively correlated (P ≤ 0.10) with average daily gain. Supplemental PROB increased (P ≤ 0.05) the relative abundance of Prevotellaceae, Megasphaera, Dorea, Acetitomaculum, and Blautia. In contrast, the relative abundance of Elusimicrobium, Moheibacter, and Stenotrophomonas were reduced (P ≤ 0.05). This study demonstrated that PROB altered the plasma carbonyl-metabolome and fecal bacterial community of the beef steers.