Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the Cd proportion in the vacuoles of protoplasts remained at a certain level (82.24%-83.40%), and the protoplast maintained its internal stable concentration (73.81-77.46 mg/L), showing the ultimate capacities for the vacuolar sequestration and cellular storage of Cd. In addition, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2=0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.