The LuxS enzyme plays a key role in both quorum sensing (QS) and the regulation of bacterial growth. It catalyzes the production of autoinducer-2 (AI-2) signaling molecule, which is a component of the methyl cycle and methionine metabolism. This study aimed at investigating the differences between the Lactobacillus rhamnosus GG (LGG) wild-type strain (WT) and its luxS mutant (ΔluxS) during biofilm formation and when resisting to inflammation caused by Enterotoxigenic Escherichia coli (ETEC) in germ-free zebrafish. Our results suggest that in the absence of luxS when LGG was knocked out, biofilm formation, extracellular polysaccharide secretion and adhesion were all compromised. Addition of synthetic AI-2 indeed rescued, at least partially, the deficiencies observed in the mutant strain. The colonizing and immunomodulatory function in WT versus ΔluxS mutants were further studied in a germ-free zebrafish model. The concentration of AI-2 signaling molecules decreased sharply in zebrafish infected with the ΔluxS. At the same time, compared with the ΔluxS, the wild-type strain could colonize the germ-free zebrafish more effectively. Our transcriptome results suggest that genes involved in immunity, signal transduction, and cell adhesion were downregulated in zebrafish infected with ΔluxS and WT. In the WT, the immune system of germ-free zebrafish was activated more effectively through the MAPK and NF-κB pathway, and its ability to fight the infection against ETEC was increased. Together, our results demonstrate that the AI-2/LuxS system plays an important role in biofilm formation to improve LGG and alleviate inflammation caused by ETEC in germ-free zebrafish. IMPORTANCE Lactobacillus rhamnosus GG is a widely used probiotic to improve host intestinal health, promote growth, reduce diarrhea, and modulate immunity. In recent years, the bacterial quorum sensing system has attracted much attention; however, there has not been much research on the effect of the LuxS/AI-2 quorum sensing system of Lactobacillus on bacteriostasis, microbial ecology balance, and immune regulation in intestine. In this study, we used germ-free zebrafish as an animal model to compare the differences between wild-type and luxS mutant strains. We showed how AI-2/LuxS QS affects the release of AI-2 and how QS regulates the colonization, EPS synthesis and biofilm formation of LGG. This study provides an idea for the targeted regulation of animal intestinal health with probiotics by controlling bacteria quorum sensing system.