In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LβT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the -304/-95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the -125/-91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.