Abstract: OBJECTIVES: To determine the association between plasma hemoglobin (HB) at three time-points (birth, postnatal days 0–3 and 0–10) and spontaneous closure of the ductus arteriosus (sDAC). STUDY DESIGN:A retrospective case-control study of preterm infants born (2013–2016) between 24 and 29 weeks of gestational age (GA) was conducted in a level three perinatal center in Switzerland. We collected hemoglobin at birth, between days 0–3 and 0–10 in two distinct groups: (i) patients treated for a PDA and (ii) patients with spontaneous closure of the ductus arteriosus (sDAC). Antenatal and postnatal demographic data and neonatal morbidity were collected. Bivariate analysis was performed and a stepwise logistic regression was done to investigate factors associated with sDAC. RESULTS: We reviewed the medical chart of 184 premature infants of whom 146 (79.3%) satisfied eligibility criteria. Of these, 74 (51%) were classified as sDAC. Patients with sDAC were older (GA: 28 vs 27, p < 0.001), more stable (clinical risk index for babies score (CRIB score): 2 vs 5, p < 0.001) and had better clinical outcomes than patients who received treatment for a PDA. Infants in the sDAC group had a higher level of hemoglobin during the first ten postnatal days. Multiple logistic regression analysis revealed that lower HB level (day 0–10) were associated with failure of sDAC (p < 0.05). CONCLUSIONS: This is one of the first studies to highlight a potential association between hemoglobin during the transitional period and sDAC. The biological nature of this observation requires prospective clarification.