This study was to explore estrogen receptor (ER) involvement in FSH and TGFβ1-stimulated steroidogenesis in rat ovarian granulosa cells. We first determined the specific involvement of ERα and ERβ in the process, and then investigated the molecular interaction of ERα and transcription coregulators in FSH and TGFβ1 up-regulation of steroidogenic gene expression. Primary culture of ovarian granulosa cells from antral follicles of gonadotropin-primed immature rats was used. Interestingly, a selective ERα antagonist methyl-piperidino-pyrazole (MPP) [like ER antagonist ICI-182,780 (ICI)] decreased FSH ± TGFβ1-stimulated progesterone production, whereas an androgen receptor antagonist hydroxyflutamide and particularly a selective ERβ antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol had no significant effect. Consistent with this, a selective ERβ agonist diarylpropionitrile (unlike 17β-estradiol) also had no effect on FSH ± TGFβ1-stimulated progesterone production. Furthermore, a selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (like 17β-estradiol) enhanced FSH-stimulated progesterone production, and this was abolished by pretreatment with MPP. Immunoblotting and chromatin immunoprecipitation analyses indicate that MPP/ICI suppression of FSH ± TGFβ1 action is partly attributed to the reduced ERα-mediated expression of Hsd3b and Cyp11a1 genes, but not steroidogenic acute regulatory protein. Furthermore, FSH ± TGFβ1 increased ERα association with histone acetylases (CBP and SRC-1) and coactivator of peroxisome proliferator-activated receptor γ (PGC-1α), and MPP/ICI dramatically reduced these interactions. In addition, FSH ± TGFβ1 increased CBP, SRC-1, and PGC-1α binding to Hsd3b and Cyp11a1 genes. Together, we demonstrate for the first time that ERα interaction with transcription coregulators, histone acetylases (CBP/SRC-1), and PGC-1α is crucial to FSH and TGFβ1-up-regulated expression of Hsd3b and Cyp11a1, and, thus, progesterone production in rat ovarian granulosa cells.