This study investigates the effect of SPIONs (superparamagnetic iron oxide nanoparticles, similar to 12.5 nm in size) on summer squash plant (Cucurbita pepo) in the presence and absence of supplementary iron (Fe(II)-EDTA). The plants were grown in nutrient solution with different iron sources: (i) Fe(II)-EDTA, (ii) without Fe(II)-EDTA (iii) SPIONs only, and (iv) Fe(II)-EDTA with SPIONs. Plant growth and development were assessed after 20 days of soaking by measuring phenological parameters such as plant biomass, chlorophyll content, amount of carotenoids, and the catalase enzyme activity. Transmission electron microscopy, inductively coupled plasma atomic emission spectroscopy, X-ray diffraction, and vibrating sample magnetometer methods were used to detect uptake and translocation of SPIONs in plant tissues. Our results showed that SPIONs treatment (without Fe(II)-EDTA) caused growth retardation and decreased the plant biomass and chlorophyll content. Hence, they are not efficient sources to compensate for iron demand of squash plant. Electron microscopy observations, magnetization and elemental analyses revealed that SPIONs are taken-up by plant roots but not translocate to upper organs. In roots, SPIONs use a symplastic route for intercellular transfer. These findings suggest that as an iron source, SPIONs alone are not efficient for plant growth, but can contribute it together with Fe(II)-EDTA.