Activin is a member of the TGFβ superfamily of growth and differentiation factors that control a variety of cellular and physiological functions. The canonical intracellular pathway of this ligand is well established and involves Smad signaling molecules. The tissue- and cell-specificity of activin action is achieved by Smad interaction with various transcriptional co-factors in the nucleus. In the reproductive axis, activin induces biosynthesis and secretion of follicle stimulating hormone (FSH) through transcriptional control of FSHβ-subunit. Whereas it has been well demonstrated that this regulation is mediated by Smad pathway, the molecular mechanisms underlying gonadotrope-specific expression of the FSHβ gene are not fully understood. Previously, we have identified Pitx2 as a pituitary-expressed transcription factor involved in activin-dependent induction of the FSHβ promoter. Present data demonstrate that Pitx2 is not only sufficient, but also necessary for FSHβ gene transcription, as a siRNA-mediated downregulation of Pitx2 protein expression abrogates both Smad3- and activin-mediated stimulation of the FSHβ promoter. In addition, downregulation of Smad3 protein expression has a significant effect on Pitx2-dependent stimulation of the FSHβ promoter, suggesting that cooperation between these factors is necessary for full transcriptional activation of the FSHβ promoter. Furthermore, we show that Pitx2/Smad protein complexes assemble and can be co-immunoprecipitated. This interaction is mediated through the homeodomain of Pitx2 and is important for stimulation of FSHβ gene transcription. Overall, these data contribute to the emerging molecular mechanism underlying both basal and activin-dependent FSHβ gene regulation.