Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.