In order to explore the mechanism of the resistance of Astragalus membranaceus seedlings to stress of copper ion (Cu²⁺) and the mitigative effects of attapulgite clay on Cu²⁺ pollution, the physiological toxicity of CuSO₄ (2~20mmol/L) to Astragalus membranaceus seedlings and the alleviative effects of attapulgite clay on such physiological toxicity were investigated. The results showed that, after treatment with 2mmol/L CuSO₄, the Cu²⁺ content, H₂O₂ (hydrogen peroxide) content, and MDA (malondialdehyde) content of roots were significantly increased by 1.82, 1.04 and, 2.14 folds, respectively, compared to the controls. After treatment with 8mmol/L CuSO₄, the SOD (superoxide dismutase) activity in the roots, the degree of damages on root apical membrane, and leaf Cu²⁺ content were significantly increased by 1.13, 1.12, and 2.62 folds, respectively, compared to the controls. Under the stress of 8mmol/L CuSO₄, the Y(Ⅱ) (photochemical quantum yield of PS Ⅱ), qP (photochemical quenching coefficient), ETR (the rate of non-cyclic electron-transport through PS Ⅱ), and chlorophyll content of the leaves significantly decreased by 22.88%, 24.44%, 21.49%, and 8.31%. While the NPQ and qN (non-photochemical quenching) of the leaves was significantly increased by 2.35 and 1.58 fold, respectively, compared with controls. The POD (peroxidase) activity, CAT (catalase) activity, and soluble protein and sugar content of roots reached maximum at 8mmol/L CuSO₄ and decreased afterwards. CuSO₄ at the concentrations as high as 15~20mmol/L caused the significant decreases of the APX (ascorbate peroxidase) activity of roots, maximum photochemical efficiency of PS II under light adaptation (F_v'/F_m') of leaves, and fresh or dry weight of whole seedlings and below-ground parts. Under the condition without CuSO₄ stress, the presence of attapulgite clay in the growth substrate made the MDA content of roots significantly decreased by 15.93% but did not affect other physiological indicators. Under the stress of 20mmol/L CuSO₄, attapulgite clay made the Cu²⁺ content in roots and leaves significantly decreased by 30.78% and 23.12%, respectively. In addition, it alleviated the effects of CuSO₄ on the production of reactive oxygen species antioxidant enzyme activities, membrane lipid peroxidation, the degree of damage on root apical membrane, and the content of soluble protein and sugar of roots. The presence of attapulgite clay in the growth also effectively alleviated the inhibition of PS II photochemical activity, chlorophyll content, and growth of the seedlings under CuSO₄ stress. These results suggested that attapulgite clay in the growth medium could effectively alleviate the CuSO₄-induced physiological toxicity to Astragalus membranaceus seedlings through reducing the bioavailability of Cu²⁺ in seedlings.