Our results show that JH is necessary for oocyte development and egg-laying in B. terrestris workers and therefore provide the strongest available support for the hypothesis that JH functions as a gonadotropin in the bumblebee B. terrestris. These findings for the bumblebee contrast with evidence that in honey bees JH does not have a similar function and highlight an evolutionary enigma relating to the role of JH signaling pathways in the evolution of sociality in bees. Our findings support and extend previous studies showing positive correlations between JH and oocyte development in B. terrestris, as well as acceleration of oocyte growth in bees treated with JHI, JHIII, or JH analogues. The current study is the first to include manipulations that reduce JH levels and thus demonstrates that JH is necessary for bumblebee reproduction. Hemolymph JH titers were strictly reduced in allatectomized bees, showing that the CA removal protocol was effective, and suggesting that that the CA glands are the only source of JH in bumblebees, as was also shown for other insects. In all conducted experiments, allatectomized bees had undeveloped ovaries containing only oocytes at basal developmental stages. This finding is notable given that the bees were kept in small queenless groups, a social Temozolomide environment in which ovarian development is typically rapid. The stronger influence of two compared to a single replacement JH treatments shows that bees was due to the lack of JH and not other factors that may have been compromised by the allatectomy surgery. Allatectomized bees also showed reduced Vg transcript levels in the fat body, and protein levels in the hemolymph; the recovery of these reductions by replacement therapy are consistent with the hypothesis that JH regulates oogenesis by activating the production of the yolk protein Vg in the fat body. The strong and opposing influences of allatectomy and replacement therapies on the fat body expression of Kr-h1 suggest that this transcription factor mediates at least part of the influences of JH on the fat body. This premise is also consistent with studies showing that JH stimulates brain Kr-h1 expression in B. terrestris and that it is a canonical component of JH signaling pathways in insects. Based on these findings we propose that in B. terrestris JH regulates oogenesis by activating Vg transcription in the fat body in a signaling pathway involving the JH-responsive transcription factor Kr-h1. Following this transcriptional activation the VG protein is released into the hemolymph and transported to the ovaries in which it is deposited in the developing oocytes. It is yet to be determined whether JH is also involved in additional processes that are necessary for oocyte development. Our study further shows that the influence of JH on bumblebee reproduction is not limited to ovary activation. Wax secretion was severly compromized in allatectomized bees, and this was partialy recuperated by replacement therapy with two JH treatments. Bumblebees use the wax they secrete for building pots and cells, including egg and brood cells and therefore, wax secretion needs to be coordinated with other reproductive activities.