Copper nanoparticles (CuNPs) are used in a range of industries such as semiconductors, catalysts, sensors, and antimicrobial agents. While there are already studies on its possible genotoxicity, few of these reports evidence in vivo.Copper nanoparticles (CuNPs) were prepared via chemical reduction and characterized by electronic transmission microscopy (TEM) and X-ray diffraction. Drosophila melanogaster (D. melanogaster) were reared on CuNPs, and Cu +2 (as CuSO4) treated food from egg to egg stage. The total number of progeny, percentage of aberrant phenotypes, oxidative stress, and gene expression of heat shock protein-70 (Hsp70) and superoxide dismutase 2 (Sod2) were investigated. Results showed that the acute exposure of CuNPs did not affect the fly's survivorship, unlike Cu +2, which showed higher toxicity. Chronic exposure of D. melanogaster to CuNPs (100 ppm) and Cu +2 (50 and 100 ppm) resulted in a delay in the development of three consecutive generations. Furthermore, the ingestion of Cu +2 and CuNPs during early developmental stages caused a dose-dependent reduction in the number of emerged flies. CuNPs and Cu +2 treatments resulted in distinctive phenotypic aberrations, such as deformed wings transmitted to the offspring in subsequent generations. Finally, CuNPs and Cu +2 treatments caused downregulation of the Sod2 gene and upregulation of the Hsp70 gene in the second and third generations. This study indicated that CuNPs are mutagenic for D. melanogaster. So, it is necessary to evaluate CuNPs toxicity to reduce human health-related issues.