The widespread use of copper oxide nanoparticles (CuO NPs) has raised concerns over their potential toxic impacts on ecosystem and human health due to their release from various products to the environment. Solanum nigrum L. is recognized as phytoremediation plant and high tolerance in the metal-stressed environment. Murashige and Skoog (MS) nutrient media supplemented with five CuO NPs levels were evaluated for their effects on the callus of S. nigrum. In excess of CuO NPs, S. nigrum tissues showed a reduction in dry weight of tissue, free amino acids, and potassium contents. We also observed increased levels of malondialdehyde (MDA) and soluble carbohydrates. In addition, Cu accumulation in callus was increased with increasing CuO NPs concentration in the medium (50-200 µg/ml).