Most insects must accumulate certain metabolites before entering diapause by virtue of photoperiodic regulation. Photoperiods influence diapause beyond the maternal generation in Cotesia vestalis (Haliday) (Hymenoptera: Braconidae), which is one of the most important larval endoparasitoid of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae). The accumulation of metabolites in the non-diapausing prepupae, adults, and eggs (before and after oviposition) in progeny from C. vestalis cultures reared at 25°C under three photoperiods (8:16, 12:12, and 16:8 (L:D) hr), along with diapausing prepupae were measured to investigate the effects of photoperiod on the accumulation of metabolites during diapause induction. Compared with those of non-diapausing prepupae, free water, trehalose, total lipid titer, and total polyol contents in diapausing prepupae showed no significant differences; but weight, bound water, total sugar, glycogen, triglyceride, and protein contents were significantly higher. Under 8 hr short days, non-diapausing prepupae and adult females were significantly heavier, and held more bound water content, however, had less free water content than those under 16 hr long days; during diapause induction from maternal generation to progeny, the variation trends of triglyceride and protein contents were irregular, such as at one stage the contents under 8 hr short lights were higher, at another stage were lower, and there was even no difference at the other stage with those under long light. Total polyol (except at prepupal stage) increased with the increase of light period, but glycogen contents increased as the light period was reduced from prepupal, adult stages to egg stages (before and after oviposition) in progeny. These results revealed that in C. vestalis, the parasitoids might start accumulating glycogen, rather than other metabolites, before entering diapause, even at maternal generation influenced by short photoperiod. Meanwhile, lower total polyol content, and higher bound water content, dry weight under short light may be prepared for surviving adverse environments (desiccation and cold) after diapause. The condition experienced by maternal generation must be concerned during diapause induction.