In the progress of inflammatory bowel disease and colitis-associated carcinogenesis, intestinal bacteria play an important role in reducing inflammatory reaction and regulating tumorigenesis. Here, we first conducted a colorectal cancer model by azoxymethane (AOM)/dextran sulfate sodium (DSS), and then the curative effects of the probiotic Lactobacillus rhamnosus Probio-M9 (Probio-M9) on gastrointestinal inflammation, colitis-associated tumorigenesis and gut microbiota in AOM/DSS mice model were conducted. Probio-M9 (approximately 2 x 109 cells/day/mouse) was administered for 3 weeks. The results showed that AOM/DSS induced higher stool consistency, increased number of colon tumors, overexpression of inflammatory cytokine, increased inflammation score and CD68+ macrophage cells in the mucosal and subserosa layer of non-tumor areas. Probio-M9 ameliorated diarrhea, inflammation and tumorigenesis. Meanwhile, Probio-M9 modified the gut microbial disorder, characterized by the regulation of the normalization of the diversity and structure of the intestinal microbiota in AOM/DSS mouse models, and increase the beneficial bacteria in intestinal tract to occupy niche of pathogenic bacteria. Thus, our findings support L. rhamnosus Probio-M9 as a promising probiotic to improve the therapeutic effects for colitis-associated carcinogenesis.