The TGF-β superfamily members are indicated to play key roles in ovarian follicular development, such as granulosa cell proliferation, estrogens, and progesterone production. However, little is known about the roles of TGF-β3 in follicular development. In this study, we found that TGF-β3 was predominantly expressed in granulosa cells of mouse ovarian follicles, and it significantly promoted 17β-estradiol (E(2)) release in a dose-dependent manner. The orphan nuclear receptor steroidogenic factor-1 (SF-1) was required in TGF-β3-induced Cyp19a1 (a key rate-limiting enzyme for estrogen biosynthesis) expression and E(2) release. Additionally, TGF-β3 enhanced the binding of SF-1 to endogenous ovary-specific Cyp19a1 type II promoter, as evidenced by chromatin immunoprecipitation assays. The enhanced effect of SF-1 by TGF-β3 may be mediated through functional interactions between SF-1 and mothers against decapentaplegic homolog (Smad)3 (a mediator of TGF-β signaling pathway), because disruption of the interaction abolished the synergistic effects of SF-1, Smad3, and TGF-β3 on Cyp19a1 mRNA expression. RNA interference and chromatin immunoprecipitation studies also demonstrated that Smad3 was required for SF-1 binding to Cyp19a1 type II promoter and activation of Cyp19a1. Smad3 thus acts as a point of convergence that involves integration of SF-1 and TGF-β signaling in affecting E(2) production. Taken together, our data provide mechanistic insights into the roles of SF-1 in TGF-β3-mediated E(2) synthesis. Understanding of potential cross-points between extracellular signals affecting estrogen production will help to discover new therapeutic targets in estrogen-related diseases.