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• Our study is firstly to investigate the effect of Clostridium butyricum on structure and composition of gut microbiota and also explore the its effect on suppression of proinflammatory cytokines and NF-κB pathway by which reduced colitis associated colon cancer in mice. • We also explore the effect of Clostridium butyricum on expression of proinflammatory cytokines such as TNF-a, IL-6 and enzyme COX-2 and to explore whether Clostridium butyricum can promote apoptosis of colorectal cancer cells by blocking the activation of Nuclear Factor-kappa B (NF-κB) pathway. • We find that CB could regulate structure and composition of gut microbiota and reduces colitis associated colon cancer in mice, the mechanism may be inhibiting NF-κB pathway and promoting apoptosis. We investigate the effects of Clostridium butyricum(CB) on gut microbiota and colitis associated colon cancer(CAC) in mice.6–8 weeks old C57BL/6 mice were randomly divided into control, azoxymethane (AOM) + dextran sodium sulphate (DSS) and AOM + DSS + CB groups. Mice in the latter two groups received an intraperitoneal injection of AOM (12.5 mg/kg), followed by three cycles of DSS diluted in water (2.5% w/v). Mice in treatment group received CB (2 × 108 CFU in 200 ul normal saline) by gavage administration three times one week. Microbiota composition was assessed by 16S rRNA high-throughput sequencing. Colon samples were collected to examine severity of colitis and tumorigenesis. Cytokines including TNF-a, IL-6 and Cyclo-oxygenase-2 (COX-2) were detected by RT-qPCR. Expression of Bcl-2, Bax and the state of components of NF-κB signaling pathway were detected by western blot. The results revealed that CB regulated structure of intestinal flora and changed the microbial composition; decreased Firmicutes/ Bacteroidetes ratio in phylum level and increased the relative abundance of probiotics; decreased colitis, decreased incidence and size of colorectal cancer(CRC) and increased apoptosis of tumor cells; decreased cytokines including TNF-a and IL-6; decreased level of COX-2; decreased phosphorylation of NF-κB; decreased level of Bcl-2 and increased expression of Bax. In conclusion, CB could regulate structure and composition of gut microbiota and reduces colitis associated colon cancer in mice, the mechanism may be inhibiting NF-κB pathway and promoting apoptosis. [ABSTRACT FROM AUTHOR]