For the first time, phosphonate-functionalized magnetite nanoparticles (Fe₃O₄ NPs) were synthesized using a one-step continuous hydrothermal process. The NP surface was modified using a hydrophilic organic molecule, namely 6-phosphonohexanoic acid (PHA). NPs were fully characterized (TEM, XRD, DLS, ζ-potential, TGA, FTIR, XPS and specific surface area measurements) in order to investigate PHA effect on size, oxidation state, anchoring and colloidal stability. PHA reduced the crystallite size and size distribution and improved greatly colloidal stability when compared with bare Fe₃O₄ NPs. Moreover, PHA was grafted on the NP surface according to three different conformations: as mononuclear monodendates, as binuclear bidentates or as lying-down complexes. This report is very promising regarding the stabilization and functionalization of Fe₃O₄ NPs by phosphonate molecules under continuous hydrothermal conditions. The post-grafting of polymers such as polyethylene glycol can be considered owing to the presence of free carboxyl groups (–COOH) on the surface of Fe₃O₄ NPs.