The recent introduction of nanotechnology in plant science has paved the way for the development of more efficient fertilizers. However, progress in research is currently hampered by a poor understanding of the interactions between nanomaterials and plant tissue. The present study investigates the potential of nano-hydroxyapatite (nHAP) as a phosphorus (P) fertilizer and the mechanisms involved during plant utilization of such nanoparticles. Using a combination of techniques to assay P functionality in metabolism and for bioimaging of P with cellular resolution, we demonstrate that contact between citrate-stabilized nHAP and roots of P deficient barley plants (grown hydroponically for 21 days) leads to a significant increase in leaf P concentration (from <2000 to >6000 ppm) and to a subsequent restoration of plant P functionality in less than 48 hours. Within the first 24 hours, intact nHAP adheres to the root epidermis without dissolving. Within the following 24 hours, nHAP penetrates the roots through the apoplast of mature epidermal and cortical cells, and dissolve here due to the acidic environment of the cell wall matrix. The mucilage layer surrounding the root cap prevents, in this specific region, the entrance of nHAP, which gradually dissolves without penetrating into deeper cell layers. In parallel, we investigate citrate-stabilized nano-rock phosphate (nRP), and demonstrate that it possesses equivalent plant bioavailability as synthetic nHAP. Since nRP was prepared through simple mechanical grinding of mined and flotated rock phosphate, this underlines its potential for direct use in agriculture, provided that contact between root tissue and nanoparticles is established. Overall, the results provide the first mechanistic evidence showing how nutrient deficient plants interact with intact nanoparticles to restore functionality in metabolism.