The antioxidant activity of selenium-containing soybean peptides (SePPs) has been previously demonstrated, despite their limited absorption in the small intestine. This study investigates the antioxidant mechanism of a selenium-containing tetrapeptide, Ser-Phe-Gln-SeM (SFQSeM), identified from SePPs, with particular emphasis on its interaction with the intestinal microbiota and its role in modulating host antioxidant defenses. The effects of SFQSeM were evaluated in a D-galactose-induced oxidative stress model and an antibiotic-treated mouse model. SFQSeM supplementation significantly reduced the oxidative stress in D-galactose-treated mice. It also promoted the growth of beneficial bacteria and increased the levels of acetate, butyrate and lactate in the intestine (P < 0.05). In the antibiotic-treated mouse model, depletion of the intestinal microbiota significantly reduced hepatic glutathione peroxidase (GSH-Px) activity (26.6%) and glutathione peroxidase 1 (GPx-1) expression (48.77%) compared to normal mice supplemented with SFQSeM (P < 0.05). In contrast to Na2SeO3 and selenomethionine, SFQSeM effectively restored the diversity of the intestinal microbiota disrupted by antibiotics. Lactobacillus, Lachnospiraceae_NK4A136_group, and Muribaculaceae were identified as predominant bacteria in the SFQSeM group, and were strongly associated with increased hepatic GSH-Px activity and GPx-1 mRNA expression (P < 0.05). In conclusion, intestinal microbiota enhances the antioxidant efficacy of SFQSeM by modulating microbial composition, producing active metabolites, and converting SFQSeM into a bioactive form of selenium.