ABSTRACTDietary iron intake is closely related to the incidence of colorectal cancer. However, the interactions among dietary iron, gut microbiota, and epithelial cells in promoting tumorigenesis have rarely been discussed. Here, we report that gut microbiota plays a crucial role in promoting colorectal tumorigenesis in multiple mice models under excessive dietary iron intake. Gut microbiota modulated by excessive dietary iron are pathogenic, irritating the permeability of the gut barrier and causing leakage of lumen bacteria. Mechanistically, epithelial cells released more secretory leukocyte protease inhibitor (SLPI) to combat the leaked bacteria and limit inflammation. The upregulated SLPI acted as a pro-tumorigenic factor and promoted colorectal tumorigenesis by activating the MAPK signaling pathway. Moreover, excessive dietary iron significantly depletedAkkermansiaceaein the gut microbiota; while supplementation withAkkermansia muciniphilacould successfully attenuate the tumorigenic effect from excessive dietary iron. Overall, excessive dietary iron perturbs diet – microbiome–epithelium interactions, which contributes to intestinal tumor initiation.