Two-component systems (TCS) are ubiquitous among bacteria, playing key roles in signalling events. However, to what extent the TCS of Rahnella aquatilis (a Phosphate solubilizing bacteria) is influenced by the hyphosphere of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis is totally unknown. Here, the expression of 16 genes encoding the TCS of R. aquatilis (i.e. involved in carbon-sensing and nutrient-sensing) and of eight genes regulated by the PhoR TCS (i.e. involved in inorganic and organic phosphorus mobilization) were analysed at regular intervals in presence of hyphae of R. irregularis. The study was conducted under in vitro culture conditions with phytate as the unique source of phosphorus. In presence of the AM fungus, the expression of TCS genes involved in carbon-sensing and nutrient-sensing were stimulated. Only, BaeS at 30 and 120 min, and BaeR at 60 min were inhibited. In addition, the PhoR TCS stimulated the expression of genes encoding phosphatase but inhibited the expression of genes involved in gluconic acid production. As the mechanism of coupling environmental changes with cellular physiological changes, TCS plays a pivotal role in regulating specific gene expression in R. aquatilis, recognizing environmental signals. More importantly, TCS genes may regulate bacteria response to hyphal carbon to mobilize phosphorus efficiently in the hyphosphere.