Objectives: The interaction between the quorum sensing (QS) molecules of gut microbiota and the immunity of colorectal cancer (CRC) has not been investigated before. Methods: We measured the concentration of autoinducer-2 (AI-2) in samples of stool, colorectal tissue, saliva and serum of CRC patients, and compared this to AI-2 levels in colorectal adenoma (AD) and normal colon mucosa (NC). To explore the activated signaling pathways involved, we utilized AI-2 extracted from Fusobacterium nucleatum to stimulate macrophages and validated these in vitro findings in human CRC tissues. Results: The AI-2 concentration in both colorectal tissue and stool of CRC patients was significantly higher when compared to that in AD and NC (all P values < .01). The AI-2 concentration along with the progression of CRC in both tissues and stools was significantly increased (P= .045，P= .0003, respectively). After AI-2 stimulation, TNFSF9 was the most significantly increased protein in macrophage cells (P < .01). TNFSF9 expression was significantly higher in CRC tissues when compared to NCs (P< .0001), which was mainly derived from macrophages in the tumor microenvironment. Moreover, AI-2 level was positively associated with CD3 + T cell numbers (P= .0462), and negatively associated with CD4/CD8 ratio (P= .0113) within CRC tissues. Conclusions: We demonstrated for the first time that AI-2 may serve as a novel marker for screening CRC in the clinic. AI-2 was associated with tumor immunity in CRCs through tumor-associated macrophages and CD4/CD8 ratio in a TNFSF9-dependent manner.