The waterlogging environment generally results in the deposition of iron plaque on plant roots, which may impact the fate of metal-based nanoparticles. Here, we investigated the influence of iron plaque on the uptake, translocation, and transformation of copper oxide nanoparticles (CuO NPs) in rice plants. The results show that the presence of iron plaque dramatically reduced the Cu contents in roots and shoots by 89% and 78% of those without iron plaque under 100 mg/L CuO NP treatment. Meanwhile, the Cu accumulation in plants was negatively related to the amount of iron plaque. X-ray absorption near edge structure (XANES) analysis demonstrated lower percentage of CuO but higher proportion of Cu(I) in shoots exposed to CuO NPs with the formation of iron plaque. Furthermore, micro X-ray fluorescence (μ-XRF) combined with μ-XANES revealed that the iron plaque in the root epidermis and exodermis consisted of goethite and ferrihydrite, which hindered the uptake of CuO NPs by roots. However, a few CuO NPs were still absorbed by roots via root hairs or lateral roots, and further translocated to shoots. But eventually, more than 90% of total Cu(II) was reduced to Cu(I)-cysteine and Cu2O in leaf veins of rice plants with iron plaque.