The development of methods aimed at activation of imagos at any point of wintering provides a compelling potential avenue to utilize bees for pollination of greenhouse crops during autumn, winter, and early spring. In this study, we tested methoprene, a juvenile hormone (JH) analogue as a chemical stimulant to end a diapause of Osmia rufa L. and enable bee activation and emergence under experimental conditions. The application of methoprene significantly reduced the emergence time of adult bees in winter months as compared to vehicle (acetone) and negative controls. Bees treated with methoprene started to emerge 3–6 days earlier than bees from acetone and control groups and finished emergence 2–6 days earlier too. Statistically significant differences were observed between methoprene and controls groups of male and female in all tested incubation periods. It was also observed that the effects of methoprene were significantly more effective in female specimens during the first 2 months of winter. Moreover, in females, methoprene improved reproductive traits such as an increase in the size of terminal oocytes. Similarly, in males, methoprene treatment resulted in a significant increase in a seminal vesicle size and dynamic elevation of spermatozoa number. Taken together, our results indicate that methoprene may play an important role in the termination of diapause, bee activation, and emergence.