Chemotaxis towards root exudates and subsequent biofilm formation are very important for root colonization and for providing the beneficial functions of plant growth-promoting rhizobacteria (PGPRs). In this study, in comparison with other root-secreted compounds, D-galactose in the root exudates of cucumber was found to be a strong chemoattractant at the concentration of 1 mu M for Bacillus velezensis SQR9. Chemotaxis assays with methyl-accepting chemotaxis proteins (MCPs) deletion strains demonstrated that McpA was solely responsible for chemotaxis towards D-galactose. Interestingly, D-galactose significantly enhanced the biofilm formation of SQR9 in an McpA-dependent manner. Further experiment showed that D-galactose also enhanced root colonization by SQR9. In addition, the secretion of D-galactose by cucumber roots could be induced by inoculation with SQR9, indicating that D-galactose may be an important signal in the interaction between plant and SQR9. These findings suggested that the root-secreted D-galactose was a signal, the secretion of which was induced by the beneficial bacteria, and which in turn induced colonization of the bacteria.