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In recent years, there has been an impressive development of nanotechnology. This has resulted in the increasing release of nanomaterials (NM) into the environment, thereby causing the risk of an uncontrolled impact on living organisms, including plants. More studies indicated the biotoxic effect of NM on plants, including crops. The interaction of nanoparticles (NP) with food crops is extremely important as they are a link to the food chain. The objective of this study was to investigate the effect of negatively charged gold nanoparticles (-) AuNP (at two concentrations; 25 µg/mL or 50 µg/mL) on barley (<i>Hordeum vulgare</i> L.) root development. Morphological, histological and ultrastructural analyses (with the use of stereomicroscope, bright filed microscope and transmission electron microscope) revealed that regardless of the concentration, (-) AuNP did not enter into the plant body. However, the dose of (-) AuNP proved to be important for the plant’s response because different morphological, histological and ultrastructural changes were observed in the treated roots. The NP treatment caused: red root colouration, a local increase in the root diameter and a decreased formation of the root hair cells (on morphological level), damage to the rhizodermal cells, vacuolisation of the cortical cells, a detachment of the cell files between the cortical cells, atypical divisions of the cells, disorder of the meristem organisation (on the histological level), the appearance of periplasmic space, numerous vesicles and multivesicular bodies, electron-dense spots in cytoplasm, alterations in the structure of the mitochondria, breakdown of the tonoplast and the plasmalemma (on the ultrastructural level).