Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine follicle-stimulating hormone subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities and, in both cases, mutation of the critical cis-element abrogated these effects. In homologous LT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C, or knocking down PITX1 or 2 expression by RNA interference in LT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in co-precipitation experiments and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Further, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.