The characterization of bacterial strains with efficient root colonization ability and the mechanisms responsible for their efficient colonization is critical for the identification and application of beneficial bacteria. In this study, we found that Burkholderia strain B23 exhibited a strong niche differentiation between the rhizosphere and rhizoplane (a niche with more abundant easy‐to‐use nutrients but stronger selective pressures compared with the tightly adjacent rhizosphere) when inoculated into the field‐grown citrus trees. Full‐length 16S rDNA amplicon analysis demonstrated that the relative abundance of B23 in the rhizoplane microbiome at 3, 5, and 9 days post‐inoculation (dpi) was always higher than that at 1 dpi, whereas its relative abundance in the rhizosphere microbiome was decreased continuously, as demonstrated by a 3.18‐fold decrease at 9 dpi compared to 1 dpi. Time‐series comparative expression profiling of B23 between the rhizoplane and rhizosphere was performed at representative time points (1, 3, and 9 dpi) through metatranscriptomic analysis, and the results demonstrated that multiple genes involved in the uptake and utilization of easy‐to‐use carbohydrates and amino acids and those involved in metabolism, energy production, replication, and translation were upregulated in the rhizoplane compared with the rhizosphere at 1 dpi and 3 dpi. Several genes involved in resistance to plant‐ and microbial competitor‐derived stresses exhibited higher expression activities in the rhizoplane compared with the rhizosphere. Furthermore, gene loci responsible for the biosynthesis of the key antifungal and antibacterial metabolites occidiofungin and ornibactin were induced, and their expression levels remained relatively stable from 3 dpi to 9 dpi in the rhizoplane but not in the rhizosphere. Collectively, our findings provide novel lights into the mechanisms underlying the root colonization of the inoculated bacterial strains and serve as a basis for the identification of strains with efficient colonization ability, thus contributing to the development of beneficial bacteria applications.