Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a ß(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1 ± 3.4 in stools and PD = 13.0 ± 0.6 in intestinal contents) than control males (PD = 7.8 ± 1.6 in stool samples and PD = 9.5 ± 1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1ß, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).