Atrial natriuretic peptide (ANP) and TGF-β play counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. We have demonstrated that ANP-cyclic GMP (cGMP)-protein kinase G (PKG) signaling inhibits TGF-β signaling by blocking TGF-β-induced nuclear translocation of mothers against decapentaplegic homolog (Smad)3 in pulmonary artery smooth muscle cells (PASMC). The current study tested the novel hypothesis that activation of the ANP-cGMP-PKG pathway limits TGF-β-induced Smad3 nuclear translocation by enhancing Smad3 binding to cytosolic anchoring proteins in isolated pulmonary artery smooth muscle cells. Cells were pretreated with vehicle or cGMP and then exposed to TGF-β1 treatment. Cytosolic fractions were isolated and immunoprecipitated with a selective anti-Smad3 antibody. Differential proteomic analysis of the cytosolic Smad3-interacting proteins by two-dimensional differential in-gel electrophoresis and mass spectroscopy followed by coimmunoprecipitation and immunostaining demonstrated that Smad3 was bound to β2-tubulin in a TGF-β1/cGMP-dependent manner: binding of Smad3 to β2-tubulin was decreased by TGF-β1 and increased by cGMP treatment. A site-directed mutagenesis study demonstrated that mutating Smad3 at Thr388, but not Ser309, two potential sites of PKG-induced hyperphosphorylation, inhibited cGMP-induced Smad3 binding to β2-tubulin. Further, luciferase reporter analysis showed that muation of T388 in Smad3 abolished the inhibitory effect of cGMP on TGF-β1-induced plasminogen activator inhibitor-1 (PAI-1) transcription. In addition, disruption of β2-tubulin with the microtubule depolymerizers nocodazole and colchicine promoted Smad3 dissociation from β2-tubulin, increased both TGF-β1-induced Smad3 nuclear translocation and PAI-1 mRNA expression, and abolished the inhibitory effects of cGMP on these processes. In contrast, the microtubule stabilizers paclitaxel and epothilone B increased cytosolic Smad3 binding to β2-tubulin and enhanced the inhibitory effect of cGMP on Smad3 nuclear translocation and PAI-1 expression in response to TGF-β1. These provocative findings suggest that sequestering Smad3 by β2-tubulin in cytosol is a key mechanism by which ANP-cGMP-PKG signaling interferes with downstream signaling from TGF-β and thus protects against pulmonary arterial remodeling in response to hypoxia stress.