Abstract: A nutrient solution culture experiment was conducted to study the effects of exogenous NO donor (sodium nitroprusside) on the subcellular distribution and chemical form of copper (Cu) in tomato seedlings under the stress of 50 μmol·L^-1 of Cu²⁺ (CuCl₂). Under this stress, the biomass and plant height of tomato seedlings decreased by 33.7% and 23.1%, respectively. Exogenous NO alleviated this inhibition effect significantly, but the Cu concentration and accumulation in the seedling organs still had a significant increase. Under the Cu stress, the Cu concentration and accumulation in the seedling organs were in the order of root > leaf > stem > petiole. Exogenous NO limited the absorbed Cu transferred from root to shoot, but could not remove this translocation. Exogenous NO increased the Cu concentration in vacuole and cell wall significantly, and decreased the Cu concentration in organelle, which lessened the damage of Cu on the regular metabolic balance in cytoplasm and increased the tolerance of organelle against Cu. Exogenous NO increased the acetic acidextractable Cu (F_HAc) in root, sodium chlorideextractable Cu (F_NaCl) in stem, F_HAc in petiole, and ethanolextractable Cu (F_E) and F_NaCl in leaf, while decreased the concentration and distribution of water-extractable Cu (F_W) in different organs, which efficiently reduced the bio-toxicity of excessive copper.