Newborn pigs with chronic hypoxia-induced pulmonary hypertension (PH) have evidence of eNOS uncoupling. In this model, we showed that therapies that promote eNOS coupling, either tetrahydrobiopterin (BH), a NOS co-factor, or L-citrulline, a NO-L-arginine precursor, inhibit PH. We wanted to determine whether co-treatment with L-citrulline and a BH compound, Sapropterin Dihydrochloride, improves NO signaling and chronic hypoxia-induced PH more markedly than either alone. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received sole treatment with L-citrulline or BH, or were co-treated with L-citrulline and BH, from day 3 through 10 of hypoxia. Catheters were placed for hemodynamic measurements and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios and NO production. In untreated hypoxic piglets, pulmonary vascular resistance (PVR) was higher and NO production and eNOS dimer-to-monomer ratios were lower than in normoxic piglets. Compared to the untreated hypoxic group, PVR was lower in hypoxic piglets co-treated with L-citrulline and BH and in those treated with L-citrulline alone, but not for those treated solely with BH. NO production and eNOS dimer-to-monomer ratios were greater for all three treated hypoxic groups compared with the untreated group. Notably, greater improvements in PVR, eNOS dimer-to-monomer ratios, and NO production were found in hypoxic piglets co-treated with L-citrulline and BH than in piglets treated with either alone. Co-treatment with L-citrulline and BH more effectively improves NO signaling and inhibits chronic hypoxia-induced PH than either treatment alone. Combination therapies may offer enhanced therapeutic capacity for challenging clinical conditions, such as chronic neonatal PH.