Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the role of G6PDH in thermotolerance of the calli from Przewalskia tangutica and tobacco (Nicotiana tabacum L.) was investigated. Results showed that Przewalskia tangutica callus was more sensitive to heat stress than tobacco callus. The activity of G6PDH and antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) in calli from Przewalskia tangutica and tobacco increased after 40 °C treatment, although two calli exhibited a difference in the degree and timing of response to heat stress. When G6PDH was partially inhibited by glucosamine pretreatment, the antioxidant enzyme activities and thermotolerance in both calli significantly decreased. Simultaneously, the heat-induced H 2 O 2 content and the plasma membrane NADPH oxidase activity were also reduced. Application of H 2 O 2 increased the activity of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium, a NADPH oxidase inhibitor, counteracted heatinduced H 2 O 2 accumulation and reduced the heat-induced activity of G6PDH and antioxidant enzymes. Moreover, exogenous H 2 O 2 was effective in restoring the activity of G6PDH and antioxidant enzymes after glucosamine pretreatment. Western blot analysis showed that G6PDH gene expression in both calli was also stimulated by heat and H 2 O 2 , and blocked by DPI and glucosamine under heat stress. Taken together, under heat stress G6PDH promoted H 2 O 2 accumulation via NADPH oxidase and the elevated H 2 O 2 was involved in regulating the activity of antioxidant enzymes, which in turn facilitate to maintain the steady-state H 2 O 2 level and protect plants from the oxidative damage.Additional key words: antioxidant enzymes, hydrogen peroxide, tobacco.