Although studies have reported numerous effects of coffee on human health, few studies have examined its specific effects on gut microbiota. This study aimed to clarify the influence of coffee and galacto-oligosaccharide (GOS) consumption on gut microbiota and host responses. After mice consumed coffee and GOS, their intestines were sampled, and the bacterial counts were measured with quantitative RT-PCR. Results showed that GOS consumption significantly increased total bacteria counts in the proximal colon. Although Escherichia coli and Clostridium spp. counts significantly decreased in the proximal colon, Bifidobacterium spp. counts increased remarkably in the same area. A bacterial growth inhibition assay was also conducted, and the results showed that E. coli growth was inhibited only by a coffee agar. Host responses were also investigated, revealing that coffee and GOS consumption remarkably increased aquaporin8 expression in the proximal colon. In conclusion, coffee has antibiotic effects, and GOS significantly decreased E. coli and Clostridium spp. counts, but increased Bifidobacterium spp. counts remarkably. Aquaporin8 expression was also increased with a mixture of coffee and GOS consumption. This is the first study to demonstrate that coffee consumption can regulate gut microbiota and increase aquaporin8, both of which are necessary for maintaining intestinal balance.