The significant anatomical changes in large intestine of germ-free (GF) mice provide excellent material for understanding microbe-host crosstalk. We observed significant differences of GF mice in anatomical and physiological involving in enlarged cecum, thinned mucosal layer and enriched water in cecal content. Furthermore, integration analysis of multi-omics data revealed the associations between the structure of large intestinal mesenchymal cells and the thinning of the mucosal layer. Increased Aqp8 expression in GF mice may contribute to enhanced water secretion or altered hydrodynamics in the cecum. In addition, the proportion of epithelial cells, nutrient absorption capacity, immune function and the metabolome of cecum contents of large intestine were also significantly altered. Together, this is the first systematic study of the transcriptome and metabolome of the cecum and colon of GF mice, and these findings contribute to our understanding of the intricate interactions between microbes and the large intestine.© 2024 The Authors.