<p>Antimicrobial peptides (APs) are increasingly used in livestock production because of their effective antibacterial properties and low risk of causing drug resistance. However, their effects on intestinal barrier function in ruminants remain relatively unexplored. This study explored the effect of composite APs on colonic microbiota and barrier integrity in <i>Holstein</i> steers. Twelve steers were randomly assigned to two groups (n = 6): a control (CK) group that received a basal diet and an AP group that received the same diet supplemented with 8&#xa0;g/ (d· head) of APs over a 270-day feeding period. After humanely killing, colonic tissue and contents were collected for analysis using histology, 16S rRNA sequencing, and metabolomics. The AP group demonstrated significantly increased mucosal thickness and mucin secretion (<i>p</i> &lt; 0.05), enhanced activities of β-glucosidase, lipase, xylanase, cellulase, and pectinase (<i>p</i> &lt; 0.01), elevated acetate, propionate, and butyrate levels (<i>p</i> &lt; 0.05), upregulated polymeric immunoglobulin receptor (pIgR) expression at both gene and protein levels (<i>p</i> &lt; 0.05), reduced α-amylase activity (<i>p</i> &lt; 0.01), and decreased density and abundance of immunoglobulin (Ig)A⁺, IgG⁺ and IgM⁺ cell clusters (<i>p</i> &lt; 0.05). Additionally, AP supplementation induced functional reorganization of the intestinal microbiota. Microbial analysis indicated increased growth of beneficial bacteria, particularly Firmicutes and Ruminococcaceae_UCG-005, but a selective decrease in potentially pathogenic microorganisms. Metabolomic analysis suggested elevated lithocholic acid levels and modified lipid metabolism in the AP group. Correlation analysis demonstrated that alterations in colonic microbiota and metabolites were associated with enhanced intestinal barrier function. Hence, AP supplementation promotes intestinal health in <i>Holstein</i> steers by modulating epithelial development and microbial community composition.</p>