Photoperiods influence diapause beyond the maternal generation in Cotesia vestalis (Haliday) (Hymenoptera: Braconidae). The sizes, 20-hydroxyecdysone (20E), and juvenile hormone III (JH III) contents in the G1 diapausing prepupae, G0 nondiapausing prepupae, adults, and G1 eggs from C. vestalis cultures reared under three photoperiods (8:16 hours light:dark, 12:12 hours light:dark, and 16:8 hours light:dark) were measured to investigate the hormones’ roles on maternal information transmission and diapause regulation. Results showed that the body size of G1 diapausing prepupae was significantly larger than that of G0 nondiapausing prepupae. The sizes of G0 prepupae, female abdomens, and G1 eggs under eight-hour light were significantly larger than those under 16-hour light. The 20E content in diapausing prepupae was significantly lower than that in nondiapausing prepupae, whereas JH III content did not show significantly difference under the same photoperiod. The 20E content in maternal prepupae, mated females and G1 eggs decreased as the light period was reduced. Juvenile hormone III contents in G0-mated females and G1 eggs (after oviposition) under three photoperiods showed no significant differences. These findings suggest that 20E may play a critical role in diapause regulation of C. vestalis, and be used as a maternal environmental message to be transmitted to the next generation.