SummaryGalactomannan oligosaccharides (GMOS) have been designated as the second generation of prebiotics. This study aimed to systematically investigate the prebiotic effects of GMOS. GMOS were obtained by the hydrolysis of guar gum (5 g L−1) using β‐mannanase (160 U g−1) for 4 h, with the molecular weight significantly decreased to 665 Da. The prebiotic activity of GMOS was verified, which displayed a great proliferation effect on Lactobacillus and Bifidobacterium. After in vitro faecal fermentation, GMOS greatly improved the production of total short‐chain fatty acids (43.62 mM), especially butyric acid (7.44 mM). Meanwhile, the relative abundance of several beneficial bacteria, including Megasphaera, Lactobacillus, and Bifidobacterium, was greatly improved to 41.15%. Besides, the total relative abundance of several typical harmful bacteria decreased to 37.80%, indicating that GMOS can regulate the composition of intestinal flora. These results provide an important scientific basis for the further use of GMOS as a prebiotic.