Abstract Background Delayed cord clamping increases placental transfusion. In vaginal deliveries higher hemoglobin concentrations are found in the second-born twin. We hypothesized it is unrelated to intertwin transfusion but to the time of cord clamping. Methods: It was a prospective cohort study of 202 women delivering twins > 32 weeks of gestation. Monoamniotic pregnancy, antenatal intertwin transfusions, fetal demise or major abnormalities were excluded from the study. The time of cord clamping depended on the obstetrician’s decision. Hemoglobin, hematocrit, and reticulocyte count were measured at birth and during the second day of life. Results At birth, hemoglobin and hematocrit levels were significantly higher in the first-born twins delivered with delayed than with early cord clamping. Higher hemoglobin and hematocrit levels were observed during the second day of life in all twins delivered with delayed cord clamping. The lowest levels were observed in twins delivered with early cord clamping. Infants delivered with delayed cord clamping were at a lower risk of respiratory disorders and NICU hospitalization. Conclusion The observed differences in Hgb concentrations between the infants in a twin pregnancy are related to cord clamping time.