Under the promotion of biodegradable chelating agents, hyperaccumulators can accumulate large amounts of heavy metals. However, in practical application, excessive accumulation of heavy metals will lead to the reduction of biomass, resulting in the reduction of heavy metal accumulation efficiency. Therefore, here we seek to investigate the dynamic process of vacuolar compartmentalization regulated by biological chelators, to provide guidance for improving the accumulation efficiency of heavy metals and alleviating the reduction of biomass. In this study, subcellular isolation was used to study the changes of enzyme activities, total cadmium (Cd), Cd2+, glutathione and reactive oxygen species (ROS) contents in protoplasts and vacuoles of leaf cells of Solanum nigrum L. The results indicate that vacuolar compartmentalization is a dynamic regulation process of continuous glutathione consumption. Under the promotion of biodegradable chelating agent N, N-bis glutamic acid (GLDA), more glutathione was induced and preferentially entered the vacuole to chelate and store Cd. Moreover, for large amounts of Cd accumulation, the insufficient supply of glutathione (GSH) preferentially maintains vacuolar homeostasis and causes redox damage outside the vacuole. These results showed that insufficient glutathione production led to the reduction of biomass. Therefore, in the practical application of GLDA, the consumption inside and outside the vacuole can be met by controlling the production of glutathione, so as to increase the efficiency of heavy metal accumulation.