Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, de-differentiated state. We previously reported that acute hypoxia represses cyclic GMP dependent-protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, -smooth muscle actin (SMA) and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to de-differentiated state and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin and MHC expression, with no effect on SMA and TSP. Treatment with 30 µM DT-3, a membrane permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, SMA, vimentin and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Over-expression of PKG in FPVSMCs by transfection with a full length PKG construct tagged with GFP (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype maker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.