Steroid hormones ecdysone (E) and 20-hydroxyecdysone (20E) are not only critical for early development in Drosophila, but are also essential in adult physiology. Particularly, ecdysteroid signaling has been shown to regulate female specific metabolic state and multiple processes of oogenesis, thus functionally equivalent to estrogenic steroids in mammals. Unlike estrogenic steroids, mainly progesterone, that are essential for ovulation, it is unknown whether ecdysteroid signaling regulates Drosophila ovulation. Our recent work demonstrated that Drosophila ovulation, resembling mammalian ovulation, involves the degradation of posterior follicle cells, the rupture of mature oocytes into the oviduct, and the formation of corpus luteum by the residual follicle cells. In addition, this rupture process is induced by the octopamine (OA) signaling in mature follicle cells that elicits an increase in intracellular calcium to activate matrix metalloproteinase 2 (Mmp2) enzymatic activity. In this study, we demonstrated that ecdysteroid signaling is operating in mature follicle cells to control ovulation. Knocking down shade (shd), encoding the enzyme for converting E to 20E, in mature follicle cells inhibits OA-induced follicle rupture ex vivo and ovulation in vivo. These defects can be rescued by ectopic expression of Shd or exogenous 20E. In addition, disruption of the ecdysone receptor (EcR) function in mature follicle cells also causes similar ovulation defects. Furthermore, we showed that ecdysteroid signaling functions upstream of calcium rise to regulate Mmp2 activity. Our data strongly suggest that 20E production in follicle cells prior to ovulation is necessary to prime mature follicles to be responsive to neuronal ovulatory stimuli, thus providing mechanistic insights into steroid signaling in ovulation and further evidence that ovulation control is largely conserved between fly and mammals.