Objective: We aimed to evaluate the effect of magnesium sulfate treatment on the risk of NEC by comparing the rate of NEC between the periods of routine use or non-use of antenatal magnesium sulfate treatment for neuroprotection in preterm deliveries less than 32 weeks of gestation. Methods: This is a retrospective cohort study of neonates who were born between 24+0 and 31+6 weeks of gestations from January 2012 to December 2016. The subjects were classified into three groups (period 1; from January 2012 to December 2013 when antenatal magnesium sulfate treatment for neuroprotection was not used, period 2; from January 2014 to March 2016 when the treatment was routinely used, period 3; from April 2016 to December 2016 when the treatment was abandoned due to its potential risk of NEC. The primary outcome was NEC and neonatal death from NEC. Results: A total of 598 neonates (270 in the period 1, 264 in the period 2, and 64 in the period 3) were included in this study. In the period 2, 160 (60.6%) neonates were exposed to antenatal magnesium sulfate, and among them 124 (77.5%) were used for neuroprotection. In the period 1 and 3, 44 (16.2%) and 9 (14.0%) neonates, respectively, were exposed to antenatal magnesium sulfate and most of them were used for tocolytics or prevention of eclampsia. The rate of NEC was not significantly different among the three periods (23.0% in the period 1, 18.2% in the period 2, and 23.4% in the period 3, P=0.346). The rates of severe NEC (grade II or III), neonatal death due to NEC, and overall neonatal death were not significantly different among the three periods. Conclusion: This study implicates that the change of antenatal magnesium sulfate treatment protocol in preterm neonates was not associated with increased risk of NEC and neonatal death due to NEC. However, further studies are needed to evaluate the long-term effect of this treatment protocol change.