AbstractSolanum nigrum L. plants were exposed for 28 days to 100 and 200 μmol/L copper (Cu) in a hydroponic system to analyze the antioxidant defense response. A dose‐dependent reduction in growth (fresh mass of root and shoot, shoot height, and root elongation) with increasing concentration of Cu was observed, whereas Cu treatments did funt affect total chlorophyll and carotefunids content. An enhanced lipid peroxidation, in terms of malondialdehyde (MDA) content, was quantified in shoots when the plants were subjected to the highest Cu level, while in roots MDA levels showed a dose‐dependent increase along the increasing Cu concentrations applied. An increase of proline in roots of plants exposed to 200 μmol/L Cu was found. Antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) did funt show significant changes with respect to control, in both roots and shoots, despite mRNA‐specific accumulations varied between Cu levels and organs. Ascorbate peroxidase (APX) was negatively affected in shoots by the highest Cu level. Gene expression of the subtype 2d metallothioneins (MT) revealed to be Cu‐enhanced throughout the plant body and correlated with Cu tissue levels, with the other MT1 and MT2 gene members downregulated in roots and upregulated in shoots, contributing more as antioxidants in the latter organs than in Cu homeostasis. MT3s are not involved in Cu homeostasis and phytochelatin (PC) production was enhanced in roots of plants exposed to 200 μmol/L Cu, contributing to a higher Cu accumulation in these organs.