Within the last years, nanogold has become more and more important in nanotechnology, for example, as catalyst or in medical applications. Its rising production, application, and disposal inevitably lead to an increased emission of gold nanoparticles (Au-NPs) in the environment. However, only little is known about the uptake and effects of Au-NPs on biota. The objective of this study was to investigate the reversibility and effects of citrate-coated Au-NP uptake into the model organism barley (Hordeum vulgare L.). For this purpose, barley seeds were cultivated in Au-NP-containing nutrient solution for 2 weeks before the seedlings were transferred into Au-NP-free media and grown for another 3 weeks. Stability of Au-NPs in the cultivation media was investigated over the 2-week exposure time. Gold content in the leaves and roots of the plants was measured after 2 weeks of exposure and after 7, 14, and 21 days of regeneration by means of total reflection X-ray fluorescence (TXRF) analysis after microwave-assisted digestion. Moreover, Au-NPs within plant material were localized by transmission electron microscopy (TEM) of ultrathin cross sections. The obtained results reveal that Au-NPs accumulate in the plant roots. Concentration-dependent effects on the uptake of macronutrients and micronutrients, as well as on biomass production of exposed plants, in particular, on root growth were observed. Even though exposed barley plants were able to regenerate to a certain extent, their root growth was permanently decreased.