Galactooligosaccharides (GOS) have been identified as beneficial prebiotics for animals and human beings. Most studies have focused on the effect of GOS on the hindgut populated with abundant microbes. However, few research studies have been conducted on the small intestine, and many results are inconsistent due to the purity of GOS, commonly mixed with monosaccharides or lactose. Therefore, pure GOS with definite structures were prepared and used in the present study to evaluate their effects on intestinal barrier function, inflammatory responses and short-chain fatty acids (SCFAs) produced in the colon of mice challenged with lipopolysaccharide (LPS). The results of 1H and 13C nuclear magnetic resonance spectral analyses indicated that the main structures of GOS with a degree of polymerization of 3 (trisaccharide) and 4 (tetrasaccharide) are [β-Gal-(1 → 6)-β-Gal(1 → 4)-β-Glc] and [β-Gal-(1 → 6)-β-Gal-(1 → 6)-β-Gal-(1 → 4)-β-Glc], respectively. The results of an in vivo study in mice showed that intragastric administration of 0.5 g per kg BW GOS attenuated intestinal barrier damage and inflammatory responses induced by LPS in the jejunum and ileum, as indicated by increasing villus height and villus-to-crypt ratio, up-regulated intestinal tight junction (ZO-1, occludin, and claudin-1) gene expression, and down-regulated pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α gene expression. Nevertheless, the protective effects of GOS on the intestinal barrier are independent of glucagon-like peptide 2. In addition, 0.5 g per kg BW GOS administration promoted the recovery of colonic acetate, propionate, butyrate, and total SCFA production reduced by LPS challenge. The obtained results provide practical evidence that pure GOS can act as protective agents for intestinal health.