Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.