A greenhouse experiment was carried out to determine plant growth, reactive oxygen species (ROS) metabolism in roots and functions of plasma membrane (PM) and tonoplast in cucumber seedlings (Cucumis sativus L. cv. Xintaimici) treated with 40 μmol L-1 CuSO4·5H2O, which were either ungrafted or grafted onto the rootstock (Cucurbita ficifolia). Cu treatment inhibited growth, induced significant accumulation of H2O2 and led to serious lipid peroxidation in cucumber roots, and the ROS-scavenging enzymes activities in grafted seedlings roots were significantly higher than that of ungrafted plants, thus less accumulation in grafted cucumber roots induced by Cu. As a result, lipid peroxidation in roots decreased. Furthermore, the activities of H+-ATPase, H+-PPase and Ca2+-ATPase in PM and/or tonoplast in grafted cucumber seedlings under Cu stress were obviously higher than that in ungrafted plants, resulting into higher ability in grafted plants to expulse the excess H+, promote the cytoplasm alkalinization, regulate the intracellular Ca2+ concentration and brought the cytoplasma concentration of free Ca2+ to extremely low level under Cu stress.