Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild to severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels was not sufficient to prevent EAO development.