Despite being fairly abundant in soils, iron (Fe) represent one of the major constraints for plants growth and productivity, due to its low solubility. The possible contribution of Plant Growth-Promoting Rhizobacteria (PGPR) to the biogeochemical cycles of mineral elements aiming at increasing the metal bioavailable fraction as well as their ability to induce and/or regulate molecular and biochemical responses in plants is still under discussion. We had previously demonstrated that the presence of the PGPR Azospirillum brasilense in the rhizosphere of cucumber plants grown in calcareous soils could increase Fe nutrition alleviating Fe chlorosis symptoms. Within the present research, we showed that the inoculation of cucumber plants with the A. brasilense induced and anticipated the activation of Fe deficiency mechanisms (i.e. Fe reduction and rhizosphere acidification) in both Fe-sufficient and Fe deficient plants, thereby enhancing the capability of plants to cope with Fe stress. These biochemical mechanisms were supported by an increased expression of the genes encoding for Fe-chelate reductase (CsFRO) and PM H+-ATPase (CsHA1) and resulted in a higher Fe uptake rate. Our findings also highlighted that the molecular and physiological responses of cucumber plants to the inoculation are strictly related to the Fe nutritional status, suggesting the possible co-existence of multiple regulation mechanisms. These results further strengthen the capability of PGPRs, like Azospirillum, to modulate Fe acquisition in plants by differently triggering genes transcription. (C) 2016 Elsevier B.V. All rights reserved. ; reserved