Metal nanoparticles (NPs) released into the aquatic environment will likely accumulate in sediment and be available for sediment-dwelling invertebrates that serve as food for other organisms, such as fish. The aim of the present study was to investigate trophic transfer of copper oxide (CuO) NPs and dissolved Cu (CuCl2) from natural sediment to the sediment-dwelling worm Tubifex tubifex, and then to the predatory fish Gasterosteus aculeatus (three-spined stickleback). Cu enriched in the stable isotope 65Cu was used to increase detection and discriminate newly added/assimilated Cu from background Cu levels. Worms were exposed to sediment spiked with 65CuCl2 or 65CuO NPs (∼20 nm) at environmentally relevant concentrations for 7 days and subsequently fed to fish for 7 days. Worms accumulated 65Cu during sediment exposure to both 65CuCl2 and 65CuO NPs (0.7 and 1.1 μg 65Cu per g dw tissue, respectively), resulting in 65Cu body burdens significantly different from control. Furthermore, significantly more 65Cu was released from the sediment into the overlying water in the 65CuO NP exposures compared to the 65CuCl2 exposures. 65Cu accumulation in fish feeding on 65CuCl2 and 65CuO NP-exposed worms was limited (intestinal tissue: 80 and 65 ng g−1 dw; fish carcass: 7 and 10 ng g−1 dw, and liver: 50 and 10 ng g−1 dw, respectively). Glutathione peroxidase (gpx) mRNA expression was significantly higher in fish feeding on 65CuCl2-exposed worms compared to 65CuO NP-exposed worms (though 65Cu tissue concentrations were similar). No significant effects were detected for the other investigated genes (ctr1, mta, gcl, gr, sod, cat, zo-1). Our results show that NP-derived Cu can enter freshwater food webs, but bioaccumulation and trophic transfer under environmentally realistic exposures is low (detectable with a tracer) and not different from that of dissolved Cu.