Iron (Fe) is an essential micronutrient for plant life and development. However, in soil, Fe bioavailability is often limited and variable in space and time, thus different regions of the same root system might be exposed to different nutrient provisions. Few studies showed that the response to variable Fe provision is controlled at local and systemic levels, albeit the identity of the signals involved is still elusive. Iron itself was suggested as local mediator, whilst hormones were proposed for the long-distance signalling pathway. Therefore, the aim of this work was to assess whether Fe, when localized in a restricted area of the root system, might be involved in both local and systemic signaling. The combination of resupply experiments in a split-root system, the use of 57Fe isotope and chemical imaging techniques allowed tracing Fe movement within cucumber plants. Soon after the resupply, Fe is distributed to the whole plant, likely to overcome a minimum Fe concentration threshold aimed at repressing the deficiency response. Iron was then preferentially translocated to leaves and, only afterwards, the root system was completely resupplied. Collectively, these observations might thus highlight a root-to-shoot-to-root Fe translocation route in cucumber plants grown on a patchy nutrient substrate.