Galacto-oligosaccharides (GOS) are prebiotics that positively affect the host's gut microbiota, which is important for the health of the host. Most previous studies focused on specific flora components (e.g. Bifidobacterium and Lactobacillus); very few have investigated the relationship between flora and metabolites. Here, we used 16S rRNA analysis and metabolomics to analyze the effect of GOS on microbiota and metabolites. Results show that the abundance of Ruminococcaceae and Oscillibacter decreased significantly in GOS-fed mice. Twenty-one metabolites, including oleic acid, arachidic acid, and behenic acid, decreased significantly in the GOS-fed mice. Fatty acid synthesis and blood triglyceride content significantly decreased in the GOS-fed mice compared with those in the control mice, suggesting that GOS may improve lipid metabolism in mice. Additionally, after three weeks of a GOS-rich diet, the mouse microbiota was significantly enriched in Alloprevotella, Bacteroides, and Parasutterella. The blood glucose level of the GOS-fed group was significantly higher than that of the control group, whereas the abundance of Coprococcus and Odoribacter (butyrate-producing bacteria) was significantly decreased. The metabolism of butyrate, known to reduce plasma glucose levels, was significantly downregulated in the GOS-fed mice, an effect potentially detrimental to the glucose metabolism of the host. This dual-omics analysis provided important information on the changes in host-microbe-metabolite interactions resulting from GOS supplementation. Our results provide evidence that GOS may improve lipid metabolism, and that long-term GOS supplementation had a detrimental effect on the host's glucose metabolism, which could be important for optimizing methods of prebiotic supplementation and developing approaches to prevent diseases using prebiotic interventions.