Antibiotic exposure has adverse effects on intestinal immunity, metabolism, and gut microbiota (GM) composition, particularly by disturbing GM composition without short-term recovery. Capsaicin, a dietary irritant, is generally avoided during antibiotic therapy, but its mechanism remains unclear. To explore the effects of capsaicin on intestinal health during antibiotic administration, we conducted experiments in specifi c pathogen free (SPF) and germ-free (GF) mice and correlation analyses using 16S rRNA sequencing and nontargeted metabolomics to explore the protective role of the intestinal biological barrier. The results showed that additional supplementation of capsaicin under antibiotic exposure did not cause serious damage to the intestine, but had potential adverse effects on the structure, function, and metabolites of GM, including increasing the abundance of opportunistic pathogens (Mucispirillum and Aeromonas), enriching metabolic pathways (arachidonic acid metabolism and lipopolysaccharide biosynthesis), and metabolites associated with colon infl ammation (N-acetylhistamine). In the absence of GM barrier, the benefi cial function of capsaicin on the intestine was weakened and even induced adverse effects, suggesting that GM may have a certain mediating mechanism in the physiological function of capsaicin.