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Summary Ectomycorrhizal fungi (EMF) play a crucial role in facilitating plant nutrient uptake from the soil although inorganic nitrogen (N) can potentially diminish this role. However, the effect of inorganic N availability and organic matter on shaping EMF‐mediated plant iron (Fe) uptake remains unclear.To explore this, we performed a microcosm study onPinus taedaroots inoculated withSuillus cothurnatustreated with +/−Fe‐coated sand, +/−organic matter, and a gradient of NH₄NO₃concentrations.Mycorrhiza formation was most favorable under conditions with organic matter, without inorganic N. Synchrotron X‐ray microfluorescence imaging on ectomycorrhizal cross‐sections suggested that the effect of inorganic N on mycorrhizal Fe acquisition largely depended on organic matter supply. With organic matter, mycorrhizal Fe concentration was significantly decreased as inorganic N levels increased. Conversely, an opposite trend was observed when organic matter was absent. Spatial distribution analysis showed that Fe, zinc, calcium, and copper predominantly accumulated in the fungal mantle across all conditions, highlighting the mantle's critical role in nutrient accumulation and regulation of nutrient transfer to internal compartments.Our work illustrated that the liberation of soil mineral Fe and the EMF‐mediated plant Fe acquisition are jointly regulated by inorganic N and organic matter in the soil.