Root colonization of beneficial rhizobacteria is critical for their beneficial effects. Quorum sensing (QS) has been reported to affect the colonization of many plant pathogens. However, how QS signals regulate root colonization of beneficial rhizobacteria is unclear. In this study, the QS signal AI-2 synthetase-encoding geneluxSwas completely deleted from the genome of the plant beneficial rhizobacteriumBacillus velezensisSQR9, and bioluminescence experiments showed that AI-2 production was blocked. Deletion ofluxSreduced biofilm formation, motility, and root colonization ofB. velezensisSQR9, while addition of exogenous AI-2 to the mutant restored this phenomenon. These results indicated that AI-2 positively affects the root colonization ofB. velezensisSQR9. This study provided new insights for enhancing the colonization of beneficial rhizobacteria.