In arthropods, it is known that ecdysteroids regulate molting, limb regeneration, and reproduction through activation of the ecdysone receptor (EcR). However, the ecdysteroid signaling pathway for promotion of ovarian development in crustaceans is still unclear. In this study, three cDNA isoforms ofEcRwere cloned from the mud crabScylla paramamosain. qRT-PCR revealed that theSpEcRmRNA was abundant in the eyestalk, ovary and epidermis. During ovarian development, theSpEcRtranscripts increased from stage I (undeveloped stage) and reached a peak at stage IV (late vitellogenic stage) before dropping to a lower level at stage V (mature stage). Meanwhile, levels of 20-hydroxyecdysone (20E) in the hemolymph, detected by HPLC-MS, displayed a similar pattern of increase with ovarian development. Results fromin situhybridization indicated thatSpEcRmRNA was present in the follicular cells during vitellogenesis. Results fromin vivoexperiments revealed that 20E at 0.2 μg/g body weight significantly stimulated the expression ofSpEcRandvitellogenin(SpVg) in female crabs during the early vitellogenic stage but not during the previtellogenic stage. This was confirmed by results fromin vitroexperiments which indicated thatSpEcRandSpVgexpression levels were significantly upregulated in early vitellogenic ovarian explants incubated with 5.0 μM 20E at 3 and 6 h but not in previtellogenic ovarian explants. Finally, results fromin vitrogene silencing experiments indicated that the expression ofSpEcRandSpVgin the ovary was significantly inhibited bySpEcRdsRNA. All these results together indicated that inS. paramamosain, 20E, andSpEcR, located in the follicular cells, play important roles in the promotion of ovarian development via regulating the expression ofSpVg.