Nanoscale zero-valent iron (nZVI) might generate positive and negative effects on plant growth, since it acts as either hazardous or growth-promotion role. It is still unclear whether such dual roles can be mediated by arbuscular mycorrhizal fungi (AMF) in plant-AMF symbiosis. We first identified that in 1.5 g kg−1 nZVI (≤1.5 g kg−1 positively), maize biomass was increased by 15.83%; yet in 2.0 g kg−1 nZVI, it turned to be declined by 6.83%, relative to non-nZVI condition (CK, p < 0.05), showing a negative effect. Interestingly, the inoculation of AMF massively improved biomass by 45.18% in 1.5 g kg−1 nZVI, and relieved the growth inhibition by 2.0 g kg−1 nZVI. The event of water use efficiency followed similar trend as that of biomass. We found that proper concentration of nZVI can positively interact with rhizosphere AMF carrier, enabling more plant photosynthetic carbon to be remobilized to mycorrhiza. The scanning of transmission electron microscopy showed that excessive nZVI can infiltrate into root cortical cells and disrupt cellular homeostasis mechanism, significantly increasing iron content in roots by 76.01% (p < 0.05). Simultaneously, the images of scanning electron microscopy showed that nZVI were attached on root surface to form an insoluble iron ion (Fe3+) layer, hindering water absorption. However, they were efficiently immobilized and in situ intercepted by extraradical hyphae in mycorrhizal-nZVI symbiosis, lowering iron translocation efficiency by 6.07% (p < 0.05). Herein, the optimized structure remarkably diminished aperture blockage at root surface and improved root activities by 30.06% (p < 0.05). Particularly, next-generation sequencing demonstrated that appropriate amount of nZVI promoted the colonization and development of Funneliformis mosseae as dominant species in rhizosphere, confirming the positive interaction between AMF and nZVI, and its regulatory mechanism. Therefore, dual effects of nZVI can be actively mediated by AMF via rhizosphere interactions. The findings provided new insights into the safe and efficient application of nanomaterials in agriculture.