Cellular processing of nanomaterials may affect their physical properties at the root of various biomedical applications. When nanoparticles interact with living cells, their spatial distribution is progressively modified by cellular activity, which tends to concentrate them into intracellular compartments, changing in turn their responsivity to physical stimuli. In this paper, we investigate the consequences of cellular uptake on the related magnetic properties and NMR relaxivity of iron oxide nanoparticles. The superparamagnetic behavior (field‐dependent and temperature‐dependent magnetization curves investigated by SQUID (Superconducting Quantum Interference Device) measurements) and nuclear magnetic relaxation dispersion (NMRD) R1 profiles of citrate‐coated maghemite nanoparticles (mean diameter 8 nm) were characterized in colloidal suspension and after being uptaken by several types of cells (tumor cells, stem cells and macrophages). The temperature‐dependent magnetization as well as the NMRD profile were changed following cellular uptake depending on the stage of endocytosis process while the field‐dependent magnetization at room temperature remained unchanged. Magnetic coupling between nanoparticles confined in cell lysosomes accounts for the modification in magnetic behavior, thereby reflecting the local organization of nanoparticles. NMR longitudinal relaxivity was directly sensitive to the intracellular distribution of nanoparticles, in line with Transmission Electron Microscopy TEM observations. This study is the first attempt to link up magnetic properties and NMR characterization of iron oxide nanoparticles before and after their cell processing. Copyright © 2012 John Wiley & Sons, Ltd.