Certain intestinal microbiota alterations appear to positively correlate with tumorigenesis of CAC due to the disruption of the balance between the host and microorganisms. It is proven that blocking MyD88 signaling can prevent colitis-associated colorectal cancer (CAC) development in mice. We are aim to reveal the role of MyD88 signaling of maintaining colonic microbiota homeostasis for preventing CAC development. We here analyzed the landscape of gut microbiome in the mice model of AOM/DSS-induced CAC with MyD88 inhibitor treatment. PCoA revealed significant reduction in Lactobacillus load and increase in Escherichia load in the mucosal microbial composition of mice with CAC, compared with normal controls (NCs). Inhibitor-treatment led to almost undetectable Proteobacteria (Escherichia) and the retention of the dominance of Firmicutes and Bacteroidota (Muribaculaceae) in the mucosa. RNA sequencing analysis identified genes were up-regulated (Hp, SAA3 and IL-1F9) and down-regulated (CYP3A44, SLC30A10, GPNMB and OTC) in Inhibitor-treated mice (vs. CAC). Meanwhile, Inhibitor-treated mice had higher percentage of MUC2-positive area in colon sections (vs. CAC, which was less than NCs) by IF staining and decreased Escherichia in the mucus layer (vs. CAC) by FISH. And intestinal microbiota from mice with MyD88 inhibitor treatment could lessen the outcome of CAC by fecal microbiota transplantation. The development of CAC was involved in the increasing and ectopic Escherichia in the decreasing colonic mucus layer. MyD88 signaling blockade may maintain the host-microbiota homeostasis by up-regulating MUC2 production, increasing probiotics and their protective effects, and inhibiting the reproduction of Escherichia.