After ovulation, ovarian 3beta-hydroxysteroid dehydrogenase type II (HSD3B2) expression increases to enhance the shift of steroidogenesis toward progesterone biosynthesis. Steroidogenic factor-1 (SF-1) is a transcription factor for several genes encoding steroidogenic enzymes. However, the level of SF-1 expression decreases in the human corpus luteum (CL) after ovulation. Liver receptor homolog-1 (LRH-1) is another member of the orphan nuclear receptor family. We hypothesize that LRH-1, rather than SF-1, plays an essential role in the regulation of corpus luteum steroidogenesis. Semiquantitative RT-PCR and real-time PCR were performed to quantify the level of LRH-1 expression and correlate with HSD3B2 level. Cell transfection, mutation analysis, and EMSA were performed to examine the role of LRH-1 in the regulation of HSD3B2. LRH-1 expression was higher in CL, compared with mature ovarian follicles. Cotransfection of granulosa cells with HSD3B2 and LRH-1 resulted in a 10-fold increase of transcription. DAX-1 inhibited LRH-1-stimulated HSD3B2, which was maintained in the presence of dibutyryl cAMP. Mutation of the either of the two putative LRH-1 binding sites, which were confirmed by EMSA, in the HSD3B2 promoter decreased LRH-1 stimulation. Our findings suggest that LRH-1 is highly expressed in CL, and it plays an essential role in the regulation of HSD3B2.