Combined resveratrol and β-hydroxy-β-methylbutyric acid (HMB) supplementation with higher protein intake (14% vs 11%) significantly enhanced jejunal barrier function in Tibetan sheep through multiple mechanisms. The combination increased beneficial Firmicutes and Bacillus bacteria while reducing harmful Proteobacteria, correlating with elevated butyric acid production. This microbial shift coincided with improved antioxidant enzyme activity, reduced inflammatory markers (IL-1β, IL-6, TNF-α), and enhanced barrier gene expression (OCLN, Muc-2, ZO-1). This research extends beyond typical resveratrol studies by demonstrating synergistic effects when combined with HMB, particularly the mechanistic link between specific bacterial populations and short-chain fatty acid production. While conducted in sheep, the findings have compelling implications for human gut health optimization, especially given resveratrol's established bioavailability and HMB's existing use in human supplementation. The study's limitation lies in its animal model, but the identified pathways—butyrate-mediated barrier enhancement and microbiome modulation—are highly conserved across mammalian species. This represents incremental but meaningful progress in understanding how targeted nutritional interventions can orchestrate beneficial gut ecosystem changes, potentially informing precision approaches to intestinal health maintenance in aging populations.