Copper oxide nanoparticles (CuO NPs) accumulating in sediment can be taken up by invertebrates that serve as prey for fish. Thus, it is likely that the latter are exposed to CuO NPs via the gut. However, to this day it is unknown if CuO NPs can be taken up via the gastrointestinal tract and if and in which tissues/organs they accumulate. To address this knowledge gap, we synthesized CuO NPs enriched in the stable isotope 65Cu and incorporated them at low concentration (5 μg 65Cu g-1 ww food) into a practical diet prepared from worm homogenate, which was then fed to Three-spined Stickleback (Gasterosteus aculeatus) for 16 days. For comparison, fish were exposed to a diet spiked with a 65CuCl2 solution. Background Cu and newly taken up 65Cu in fish tissues/organs including gill, stomach, intestine, liver, spleen, gonad and carcass and feces were quantified by ICP-MS. In addition, expression levels of genes encoding for proteins related to Cu uptake, detoxification and toxicity (ctr-1, gcl, gr, gpx, sod-1, cat, mta and zo-1) were measured in selected tissues using RT-qPCR. The obtained results showed that feces of fish fed 65CuO NP-spiked diet contained important amounts of 65Cu. Furthermore, there was no significant accumulation of 65Cu in any of the analyzed internal organs, though 65Cu levels were slightly elevated in liver. No significant modulation in gene expression was measured in fish exposed to 65CuO NP-spiked diet, except for metallothionein, which was significantly upregulated in intestinal tissue compared to control fish. Altogether, our results suggests that dietary absorption efficiency of CuO NPs, their uptake across the gastrointestinal barrier into the organism, and effects on Cu-related genes is limited at low, environmentally relevant exposure doses (0.2 μg 65Cu -1 fish ww day-1).