The uptake, transport, and toxicity mechanisms of zinc oxide (ZnO) engineered nanomaterials (ZnO-ENMs) in aquatic plants remain obscure. We investigated ZnO-ENM uptake and phytotoxicity in Phragmites australis by combining Zn stable isotopes and microanalysis. Plants were exposed to four ZnO materials: micron-size ZnO, nanoparticles (NPs) of <100 nm or <50 nm, and nanowires of 50 nm diameter at concentrations of 0–1000 mg l−1. All ZnO materials reduced growth, chlorophyll content, photosynthetic efficiency, and transpiration and led to Zn precipitation outside the plasma membranes of root cells. Nanoparticles <50 nm released more Zn2+ and were more toxic, thus causing greater Zn precipitation and accumulation in the roots and reducing Zn isotopic fractionation during Zn uptake. However, fractionation by the shoots was similar for all treatments and was consistent with Zn2+ being the main form transported to the shoots. Stable Zn isotopes are useful to trace ZnO-ENM uptake and toxicity in plants.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement NANOREM No 704957. Franck Poitrasson is funded by the French Centre National de la Recherche Scientifique.