Abstract Aim: To determine if the NADPH oxidase isoform Nox4 contributes to increased H2O2 generation in persistent pulmonary hypertension of the newborn (PPHN) pulmonary arteries (PA), and to identify downstream signaling targets of Nox4 that contribute to vascular remodeling and vasoconstriction. Results: PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs, PA, and PA smooth muscle cells (PASMC) were isolated from control and PPHN lambs. Increased expression of p22(phox) and Nox4 in PPHN lungs, PA, and PASMC was associated with increased reactive oxygen species in PPHN PA, increased protein thiol oxidation in PPHN PASMC, and a decreased activity of extracellular superoxide dismutase (ecSOD) in the lungs and PASMC. Nox4 small interfering RNA (siRNA) decreased Nox4 expression and thiol oxidation and increased the ecSOD activity in PPHN PASMC. An increased activity of nuclear factor-kappa B (NFkappaB) and expression of its target gene cyclin D1 were detected in PPHN lungs, PA, and PASMC. Nox4 siRNA and catalase attenuated these increases in PASMC, and catalase decreased cyclin D1 expression in PPHN lungs. Innovation: This study demonstrates for the first time that Nox4 expression is elevated in a lamb model of neonatal pulmonary hypertension. It identifies increased NFkappaB and cyclin D1 expression and a decreased ecSOD activity as targets of increased Nox4 signaling. Conclusion: PPHN increases p22(phox) and Nox4 expression and activity resulting in elevated H2O2 levels in PPHN PA. Increased H2O2 induces vasoconstriction via mechanisms involving ecSOD inactivation, and stimulates vascular remodeling via NFkappaB activation and increased cyclin D1 expression. Approaches that inhibit the pulmonary arterial Nox4 activity may attenuate vasoconstriction and vascular remodeling in PPHN. Antioxid. Redox Signal. 18, 1765-1776.