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Adult reproductive diapause is an adaptive strategy under adverse environments for insects and other arthropod species, including bumblebees, which enables queens to survive through a harsh winter and then build new colonies in the following spring. Little research has been done on the molecular regulatory mechanism of reproductive diapause in Bombus terrestris, which is an important pollinator of wild plants and crops. Our previous research identified the conditions that induced reproductive diapause during the year-round mass rearing of B. terrestris. Here, we performed combined transcriptomics and proteomics analyses of reproductive diapause in B. terrestrisduring and after diapause at three different ecophysiological phases, diapause, postdiapause, and founder postdiapause. The analyses showed that differentially expressed proteins/genes acted in the citrate cycle, insect hormone biosynthesis, insulin and mTOR signaling pathway. To further understand the mechanisms that regulated the reproductive diapause, genes involved in the regulation of JH synthesis, insulin/TOR signal pathway were determined. The BtRheb, BtTOR, BtVg, and BtJHAMThad lower expression levels in diapause queens. The JH III titer levels and the activities of the metabolic enzymes were significantly up-regulated in postdiapause queens. Also, after the microinjection of insulin-like peptides (ILPs) and JH analogue (JHA), hormones, cold-tolerance metabolites, metabolic enzymes, and reproduction showed significant changes. Together with results from other related research, a model of the regulation of reproductive diapause during the year-round mass rearing of B. terrestriswas proposed. This study contributes to a comprehensive insight into the molecular regulatory mechanism of reproductive diapause in eusocial insects.