Silver nanoparticles (Ag-NPs) are used in a wide range of everyday products, leading to increasing concerns regarding their accumulation in soils and subsequent impact on plants. Using single particle inductively coupled plasma mass spectrometry (spICP-MS) and synchrotron-based techniques including X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM), we characterized the uptake, speciation, and translocation of insoluble AgS-NPs (an environmentally-relevant form of Ag-NPs in soils) within two plant species, a monocot and a dicot. Exposure to 10 mg Ag L as AgS-NPs for one week resulted in a substantial increase in leaf Ag concentrations (3.8 to 5.8 μg Ag g dry mass). Examination using XAS revealed that most of the Ag was present as AgS (>91%). Furthermore, analyses using spICP-MS confirmed that these AgS particles within the leaves had a markedly similar size distribution to those supplied within the hydroponic solution. These observations, for the first time, provide direct evidence that plants take up AgS-NPs without a marked selectivity in regard to particle size and without substantial transformation (dissolution or aggregation) during translocation from roots to shoots. Furthermore, after uptake, these AgS-NPs reduced growth, partially due to the solubilisation of Ag , which resulted in an upregulation of genes involved in the ethylene signalling pathway. Additionally, the upregulation of the plant defense system as a result of AgS-NPs exposure may have contributed to the decrease in plant growth. These results highlight the risks associated with Ag-NP accumulation in plants and subsequent trophic transfer the food chain.