Phenotypic differentiation of adventitial fibroblasts to myofibroblasts is an essential feature of vascular remodeling. Here, we carried out perivascular gene transfer of dominant-negative N19RhoA to investigate whether antagonism of RhoA signaling attenuates neointimal formation following rat carotid artery balloon injury and alters TGF-beta1-Smad2-induced differentiation of adventitial fibroblasts to myofibroblasts. Perivascular delivery of an adenovirus coexpressing dominant-negative N19RhoA and humanized Renilla green fluorescent protein (hrGFP) (Ad-N19RhoA-hrGFP), as demonstrated by hrGFP staining, suppressed neointimal formation at 7 and 14days post-injury. Ad-N19RhoA-hrGFP administration inhibited neointimal alpha-smooth muscle-actin and Calponin expression, as markers of myofibroblast differentiation and perivascular collagen deposition, at 14days after balloon injury. Ad-N19RhoA-hrGFP administration also inhibited adventitial Smad2 phosphorylation, but did not alter local TGF-beta1 and total-Smad2 expression after injury. Our results provide evidence that perivascular gene transfer of dominant-negative N19RhoA blocks TGF-beta1-Smad2-induced differentiation of adventitial fibroblasts to myofibroblasts, which contributes to intimal hyperplasia after balloon injury.