The toxicity of CuO nanoparticles (CuO NPs) to human hepatocarcinoma (HepG2) cells was investigated in this study. CuO NPs (1 - 40 mg/L) had significant toxicity to HepG2 cells. The antioxidant N-acetyl-L-cysteine (NAC) significantly reduces the cytotoxicity induced by the CuO NPs, supporting the hypothesis that oxidative stress contributes to the cytotoxicity of CuO NPs. To further explore the oxidative mechanisms of cytotoxicity, we examined CuO NPs-induced production of reactive oxygen species (ROS) in HepG2 cells. CuO NPs generated intracellular ROS in HepG2 cells in a concentration-dependent manner.