Aims: Magnesium sulfate has neuroprotective effects in preterm infants. Whether other antepartum treatments interfere with the neuroprotective actions is not well known. This study aims to explore the impacts of antenatal administration of Magnesium sulfate or beta-2 adrenergic agonists as tocolytic agents on the developing brain in premature infants. Methods: This is a retrospective cohort study in four tertiary perinatal centers in Japan. We collected data of pregnant women and infants born between 28 and 36 weeks for tocolytic agents, gestational age, sex, antenatal corticosteroid, fetal growth restriction, pathological chorioamnionitis, low umbilical arterial pH values (<7.1), multiple pregnancy, mode of delivery and institutions after excluding clinical chorioamnionitis, non-reassuring fetal status or major anomalies. Tocolytic agents were categorized into four groups: no-tocolysis, magnesium sulfate, beta-2 adrenergic agonists and the combination of them. We conducted multiple comparisons with multivariate analyses using generalized linear regression models to compare the prevalence of a poor perinatal outcome defined as infant's death, brain damage, particularly cerebral palsy and developmental delay. Results: Among 1083 infants, 39% were no-tocolysis, 47% were magnesium sulfate, 41% were beta-2 adrenergic agonists and 27% were combination group, including the duplication. The incidence of poor perinatal outcome was decreased by magnesium sulfate (OR 0.27, 95% CI 0.10-0.72), but not changed significantly by beta-2 adrenergic agonists (OR 1.28, 95% CI 0.63-2.59) or the combination group (OR 2.24, 95% CI 0.67-7.54), compared with the no-tocolysis. Conclusion: The combination therapy for tocolysis with beta-2 adrenergic agonists diminished the magnesium sulfate neuroprotective action after adjusting for covariables.