Activin A alters the characteristics of human arterial smooth muscle cells (SMCs) toward a contractile, quiescent phenotype. We hypothesize that activin A may prevent SMC-rich neointimal hyperplasia. Here, we study the effect of adenovirus-mediated expression of activin A on neointima formation in vitro and in vivo. Human saphenous vein organ cultures, in which a neointima is formed spontaneously, were infected either with activin A- or lacZ-adenovirus. Activin A-overexpression reduces neointima formation by 78%, whereas no significant reduction was observed after control infection. In addition, the effect of activin A on neointima formation was assessed in vivo in mice with cuffed femoral arteries. In activin A adenovirus-infected mice (IV injection), neointimal hyperplasia is reduced by 77% compared with the SMC-rich neointima in mock-infected or in noninfected mice. Cultured human saphenous vein SMCs and murine aorta SMCs were incubated with activin A and an increased expression of SM22alpha and SM alpha-actin mRNA, and SM alpha-actin protein was demonstrated. Laser-capture microdissection on sections of cuffed murine arteries and subsequent real-time RT-PCR established in vivo induction of SM alpha-actin mRNA in the media of activin A-treated mice. In summary, activin A inhibits neointima formation in vitro and in vivo by preventing SMC dedifferentiation.