The impact of plant microbiota on the health and physiology of their host is increasingly studied and recognized. However, in the rhizosphere, the functions of most bacteria and the genetic determinants involved in the molecular dialogue between plant and bacteria are poorly understood. Agrobacteria are ubiquitous soil borne and rhizospheric bacteria able to establish commensal or even beneficial interactions with plant roots. The genomic speciesAgrobacterium fabrumharbor seven specific-regions (SpG8-1 to SpG8-7), whose annotation seems to indicate a close connection with the plant during plant-bacteria interactions. To evaluate the involvement ofA. fabrum-specific regions in the plant-bacteria interaction, deletion mutant strains of eachA. fabrum-specific region were inoculated onMedicago truncatularoots. Root metabolite profiles were compared by UHPLC-UV/DAD-ESI-MS QTOF analyses, and the highlighted discriminating metabolites were annotated by tandem mass spectrometry. Metabolomic analyses have shown thatA. fabruminoculation modulates the content of phenolic compounds inM. truncatularoots, in particular flavonoids. These root metabolite modulations observed with the wild-type strain often appear to be linked to at least one of theA. fabrum-specific genes, as almost allA. fabrum-specific regions showed an influence on one or more of these specialized root metabolites. In addition, our results underlined a putative cross-talk or coordinated effect of theA. fabrum-specific regions during the interaction ofA. fabrumwithM. truncatula, as all mutants except one induced similar modifications on flavonoids. These results contribute to a better understanding of the ecological niche construction ofA. fabrumhighlighting the importance of its specific genes in the establishment of this fine-tuned interaction.