Objective: Dysbiosis of intestinal microbiome is an important colorectal cancer (CRC) pathogenetic mechanism. Lactobacillus-based probiotic cocktail could inhibit colitis-associated cancer (CAC) by alleviating intestinal dysbiosis. The intestinal microbial metabolites have been linked with CRC etiology. However, the link between Lactobacillus-based probiotic cocktail and the alteration of intestinal metabolism and theirfunctional mechanisms during CAC process is still poorly understood. Methods: For assessing protective effects of the probiotic cocktail, azomethanes/dextran sodium sulfate (AOM/DSS) induced CAC mice were pretreated with the probiotic cocktail. Colon of C57BL/6 mice were used to assess inflammation and tumorigenesis. Comparative analysis was performed for determining how the probiotic altered intestinal metabolism and gene expression. Meanwhile, intestinal microbiota alterations were analyzed. The concluding integrated analysis of intestinal metabolism and gene expression as well as intestinal microbiota was presented. Results: Pretreatment with the probiotic alleviated intestinal inflammation and limited the formation of tumors. Oncogenes were down-regulated and cancer suppressor genes were up-regulated after probiotic pretreatment. Pretreatment with the probiotic induced a rise of Lactobacillus-dominated genera and a reduction of potential pathogenic bacteria Parasutterella, Helicobacter and Muribaculum, and affected expression of intestinal metabolites that involved 37 metabolic pathways. Lactobacillus-associated intestinal metabolite variations involve five metabolic pathways-arginine and proline metabolism, histidine metabolism, pyrimidine metabolism, purine metabolism, and tyrosine metabolism. Conclusions: Pretreatment with Lactobacillus-based probiotic cocktail protected mice from CAC by interfering with intestinal metabolites that affected the cancer suppressor genes and oncogenes' expression. Furthermore, Lactobacillus affected five metabolite pathways, which was important mechanism for probiotic anti-inflammatory and anti-tumorigenesis.