Liver receptor homolog-1 (LRH-1) is a conserved member of the NR5A subfamily in vertebrates and a potential regulator of estrogen synthesis in the ovarian granulosa cells. An Lrh-1 homologue was obtained from the orange-spotted grouper Epinephelus coioides that contains the conserved structural features of NR5A and is phylogenetically closely related to NR5A2. The expression of the orange-spotted grouper Lrh-1 is tissue-specific with relatively higher levels in the liver and ovary. The immunoreactive signals for Lrh-1 and Cyp19a1a were present in the ovarian follicular cells and germ cells. In the ovarian follicular cells, Lrh-1 was present both in the nucleus and cytoplasm, and colocalized with Cyp19a1a. The expression levels of both increased during vitellogenesis whereas only Cyp19a1a dramatically decreased toward maturation when Lrh-1 was localized almost exclusively to the cytoplasm of the follicular cells. The orange-spotted grouper Lrh-1 could up-regulate cyp19a1a transcription in vitro via the two conserved Ftz-f1 sites in cyp19a1a promoter. Chromatin immunoprecipitation analysis showed that the orange-spotted grouper Lrh-1 could bind cyp19a1a promoter in vivo with a higher abundance in the vitellogenic ovary, whereas the binding was dramatically decreased in the mature ovary. Taken together, the results of present study demonstrate that Lrh-1 plays an important role in up-regulating cyp19a1a gene in the ovarian follicular cells during vitellogenesis, and the sequestration of Lrh-1 to the cytoplasm may down-regulate cyp19a1a expression in the mature ovary. This mechanism for modifying transcriptional roles of the orange-spotted grouper Lrh-1 may shed new light on the regulation of Cyp19a1 expression in other vertebrates as well.