To probe the physiological and biochemical tolerance mechanisms in Astragalus neo-mobayenii Maassoumi, an endemic plant around the Cu-rich areas from the North West of Iran, the effect of different copper concentrations at toxic levels on this plant was investigated. Copper was applied in the form of copper sulfate (CuSO₄·5H₂O) in four levels (0, 50, 100, and 150μM). We observed no visible symptoms of Cu toxicity in this plant species. During the exposure of plants to excess copper, the antioxidant defense system helped the plant to protect itself from the damage. With increasing copper concentration, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities increased in leaves and roots () compared with that of the control group. The chlorophyll amount gradually declined with increasing Cu concentrations. However, reduction in the 50μM level showed insignificant changes. Enhanced accumulation of proline content in the leaves was determined, as well as an increase of MDA content (oxidative damage biomarker) (). The results indicated that Cu contents in leaves and roots enhanced with increasing levels of Cu application. The Fe and Mn contents in both shoots and roots significantly decreased with increasing Cu concentration. Finally, the mechanisms of copper toxicity and copper tolerance in this plant were briefly discussed.