[Objectives]  To investigate changes in key biochemical substances during different stages of the diapause of Aphidoletes aphidimyza in order to better understand the metabolic characteristics of this species and physiological mechanism regulating diapause in insects in general. [Methods]  The amount of key metabolites (total protein, total carbohydrates, glycogen, trehalose and glycerin) and fresh body weight, were measured in non-diapausing, diapausing and post-diapausing A. aphidimyza (diapause induced mature larvae, diapausing prepupae and post-diapause adults). [Results]  (1) The fresh weight and amount of biochemical substances in diapause induced mature larvae were significantly higher than those in non-diapausing individuals. (2) Trehalose and glycerol significantly increased in diapausing prepupae; in 60 day-old diapausing prepupae these were 1.80 times and 1.73 times, respectively, the levels of these substances in non-diapausing prepupae. (3) The fresh weight, and trehalose and glycerin levels of post-diapause adults were significantly higher in those of non-diapausing adults, whereas the opposite was true for glycogen levels. (4) Total carbohydrate and glycogen levels gradually decreased with diapause stage, from diapause-induced mature larvae to post-diapause adults. Total protein content fluctuated, declining from diapause-induced mature larvae to the anaphase of diapausing prepupae, before rising in post-diapause adults. The opposite trend was observed for trehalose and glycerin. [Conclusion] These results indicate that diapausing prepupae accumulate trehalose and glycerin to meet the energy requirements of diapause, and to protect them from environmental stress.