KEY MESSAGE : Two soybean cultivars showed markedly different drought tolerance. G6PDH plays a central role in the process of H ( 2 ) O ( 2 ) regulated GR, DHAR, and MDHAR activities to maintain GSH and Asc levels. Glucose-6-phosphate dehydrogenase (G6PDH) plays a pivotal role in plant resistance to environmental stresses. In this study, we investigated the role of G6PDH in modulating redox homeostasis under drought stress induced by polyethylene glycol 6000 (PEG6000) in two soybean cultivars JINDOU21 (JD-21) and WDD00172 (WDD-172). The G6PDH activity markedly increased and reached a maximum at 96 h in JD-21 and 72 h in WDD-172 during PEG6000 treatments, respectively. Glucosamine (Glucm, a G6PDH inhibitor) obviously inhibited G6PDH activity in both soybeans under PEG6000 treatments. After PEG6000 treatment, JD-21 showed higher tolerance than WDD-172 not only in higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), but also in higher content of glutathione (GSH) and ascorbate (Asc). And we found that hydrogen peroxide (H(2)O(2)) regulated the cell length in root elongation zone. Diphenylene iodonium (DPI, a plasma membrane NADPH oxidase inhibitor) counteracted the PEG6000-induced H(2)O(2) accumulation and decreased the activities of GR, DHAR, and MDHAR as well as GSH and Asc content. Furthermore, exogenous application of H(2)O(2) increased the GR, DHAR, and MDHAR activities that were decreased by Glucm under drought stress. Western blot analysis showed that the G6PDH expression was stimulated by PEG6000 and buthionine sulfoximine (BSO, glutathione biosynthesis inhibitor), and blocked by Glucm, DPI and N-acetyl-L-cysteine (NAC, GSH precursor) in both cultivars. Taken together, our evidence indicates that G6PDH plays a central role in the process of H(2)O(2) regulated GR, DHAR, and MDHAR activities to maintain GSH and Asc levels.