Hydrogen peroxide is a toxic metabolite of oxidative metabolism, but may also serve as a broad spectrum signaling molecule controlling a variety of essential physiological processes. Photoperiod influences diapause beyond the maternal generation in Cotesia vestalis and we speculated that intracellular hydrogen peroxide was a possible signal mediating this generational effect. To investigate the signaling functions of hydrogen peroxide in diapause induction in C. vestalis, hydrogen peroxide and activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were measured at diapause, and in maternal non-diapausing prepupa, adult, and offspring egg stages form the cultures reared at 25°C under the photoperiods of 8∶16, 12∶12, and 16∶8 (L∶D) h. The hydrogen peroxide contents and activities of superoxide dismutase and peroxidase in maternal non-diapausing prepupae and adults declined as the light period was reduced, whereas catalase activity increased significantly. Compared to diapausing prepupae, maternal non-diapausing prepupae under 8 h short light had significantly higher superoxide dismutase, lower catalase, and similar peroxidase activities. Under the three photoperiods, the variation trends of protective enzyme activities in offspring eggs were similar to the trends in maternal prepupae and adults. The variation trends of hydrogen peroxide in eggs were opposite to the trends in maternal prepupae and adults. Peroxidase and catalase activities in eggs were significantly higher after oviposition than before oviposition. These findings indicate that hydrogen peroxide plays an important role in signal transduction between two generations.