Background: due to widespread applications of nanoparticles in biomedicine and biotechnology and their effects on human health, the present study was conducted to determine the cytotoxic effect of CuO nanoparticles on serum biochemical parameters and liver histopathological changes of NMRI mice. Methods: In this experimental study, forty two female NMRI mice were randomly divided into six groups (n=7): four experimental groups, one sham group and one control group. The experimental mice on days 3 and 12 of pregnancy received CuO nanoparticle with concentrations 300, 400, 500, 600 mg/kg intraperitoneally. After mentioned treatment, blood samples were collected and serum biochemical parameters were analyzed. On day 17 of pregnancy, mice were dissection and liver for histopathological evaluation were stained with hematoxylin and eosin. Results: Histopathological studies demonstrated dose-dependent changes in mice liver treated with CuO nanoparticles compared to control group. Some changes include congestion, necrosis, inflammatory cell infiltration, degeneration vacuoles were observed after exposure to different doses of CuO nanoparticles. The serum levels of ALT, AST, ALP and LDH enzymes increased significantly in a dose dependent manner as compared to the control group. Conclusion: The results of this study demonstrate that exposure to different doses of CuO nanoparticles can lead to irreversible damage to the liver and increased serum enzymes in NMRI mice.