Background: Inflammation plays an important role in neonatal hypoxia-induced organ damage. Newborns with perinatal asphyxia often develop persistent pulmonary hypertension of the newborn (PPHN). The objective of this study was to explore changes in the pro-inflammatory high mobility group box-I (HMGB1) protein during hypoxia-induced PPHN clinically and in vivo. Methods: Serum samples were collected from full-term newborns at PPHN onset and remission. As controls, blood serum samples were collected from the umbilical arteries of healthy full-term newborns born in our hospital during the same period. Clinical data for neonates were collected and serum levels of HMGB1, IL-6, and TNF-alpha were detected by enzyme-linked immunosorbent assay (ELISA). An animal study compared a PPHN Sprague-Dawley rat model to healthy newborn control rats. Histopathology was used to evaluate changes in the pulmonary artery wall. ELISA and western blot analyses were used to examine HMGB1 levels in the serum and lungs. Results: Serum HMGB1 levels were significantly elevated in newborns with PPHN, compared to those in healthy controls, and decreased dramatically after PPHN resolution. HMGB1 changes were positively correlated with serum tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) levels. Histopathological analysis demonstrated that the median wall thickness of pulmonary arterioles accounting for the percentage of pulmonary arteriole diameter (MT%) was not significantly different between PPHN and control groups 3 d after PPHN, although thickness of the small pulmonary arterial wall middle membrane and stenosis of the small pulmonary arteries. ELISA and western blot analyses showed similar trends between serum HMGB1 levels and HMGB1 protein expression in the lungs. Serum and lung HMGB1 levels were significantly elevated soon after PPHN onset, peaked after 24 h, and then decreased after 3 d, although they remained elevated compared to those in the control group. Conclusions: This study indicates that HMGB1 is related to hypoxia-induced PPHN pathogenesis. HMGB1 changes might thus be used as an early indicator to diagnose hypoxia-induced PPHN and evaluate its improvement. We also provide important evidence for the involvement of inflammation in the progression of hypoxia-induced PPHN.