The widespread use of copper oxide nanoparticles (CuONPs) has dramatically increased their concentrations in soils and severely affected the health of soil organisms. The gut microbiota critically contributes to the metabolism and immune system of its host and is sensitive to environmental pollution. The toxic effect of CuONPs on the gut microbiota, especially in soil fauna, still needs further research. In the present study, a comprehensive toxicological test was performed to reveal the effects of CuONPs and their metal counterpart on the gut microbiota of soil collembolans using Illumina high throughput sequencing. Furthermore, the concomitant changes in the collembolans gut-associated antibiotic resistance genes (ARGs) and metabolism were investigated using high-throughput quantitative PCR and carbon and nitrogen stable isotope compositions. Both CuONPs and ionic copper (Cu) exposure disturbed the collembolan gut microbial community structure while only CuONPs reduced the gut microbial diversity. A total of 66 ARGs were detected in the collembolan guts, and CuONPs exposure induced a reduction in both diversity and abundance of ARGs. Additionally, CuONPs and ionic Cu exposure altered the C and N stable isotope compositions of the collembolans, indicating a change in their metabolism. Moreover, structural equation modeling indicated that 85.5% of the carbon stable isotope variations and 73.3% of the nitrogen stable isotope variations were explained by changes in Cu bioaccumulation and the gut microbiota. The results of the present study extend our knowledge regarding the comprehensive toxicity of metal oxide NPs on soil fauna. (C) 2020 Elsevier Ltd. All rights reserved.