Summary To elucidate whether Juvenile Hormone (JH) regulates physiological status in relation to diapause and cold hardiness, we investigated various physiological and biochemical changes in diapause adults of Aulacophora nigripennis after treatment with exogenous pyriproxyfen, an analogue of JH. Topical application of pyriproxyfen at high dose (≥0.05 μg) caused the diapause adults not only to consume more oxygen and mate earlier, but also to suppress enhancement of chill tolerance at 0°C and accumulation of myo-inositol. By contrast, a lower dose of pyriproxyfen (0.01 μg) did not, suggesting that the amount of pyriproxyfen between 0.01 and 0.05 μg is a critical dose breaking both diapause and cold hardening. Pyriproxyfen-treated post-diapause adults lost triacylglycerols, but not glycogen, more rapidly than the acetone control. It is suggested that triacylglycerols are the main energy resource in both diapause and post-diapause adults, but are consumed more by the latter because of their higher metabolic rate. Glycogen may be converted mainly into myo-inositol during diapause, but consumed as an energy fuel for basal metabolism during post-diapause development. Thus, various physiological and biochemical traits in relation to diapause and cold tolerance in this beetle may be at least in part under the control of JH.