<p>The optimal FiO₂ threshold for surfactant administration in preterm neonates with respiratory distress syndrome (RDS) remains uncertain, with limited evidence supporting current guideline recommendations. The objective was to compare the total duration of respiratory support between two FiO₂ thresholds for surfactant administration in preterm neonates stabilized on CPAP. In this non-inferiority randomized control trial (RCT), preterm neonates (26–32 weeks’ gestation) with RDS were randomized at 1 h of life to receive surfactant at FiO₂ thresholds of either 40% or 30%. The primary outcome was total duration of respiratory support. The secondary outcomes were requirement of surfactant within 6 h after birth, requirement of repeat dose of surfactant, common morbidities of prematurity bronchopulmonary dysplasia (BPD) stage ≥ 2, air leaks, hemodynamically significant patent ductus arteriosus (hsPDA) (requiring treatment), all-cause mortality, and total duration of hospital stay. Subgroup analysis for gestation between 26 and 29 weeks was done. A total of 205 neonates with a mean birth weight of 1237.92 ± 328.89 g and a mean gestation age of 30.06 ± 1.85 weeks were enrolled. The mean duration of respiratory support was 140.8 h in 40% FiO₂ and 153.9 h in 30% FiO₂ (<i>p</i> = 0.846). Surfactant was administered to 32% and 45% neonates in 40% FiO₂ and 30% FiO₂, respectively (<i>p</i> = 0.064). Need for repeat dose of surfactant was not significantly different in the two groups (1.9% % and 7%, respectively) (<i>p</i> = 0.095). Ventilator-free days were similar between the two groups (26 days vs 25 days). Subgroup analysis (26–29 weeks) showed a reduction in mechanical ventilation duration by 33 h in the 40%FiO₂.</p><p> <i>Conclusion</i>: This first randomized controlled trial directly comparing FiO₂ thresholds of 40% versus 30% for surfactant administration in preterm infants (26–32 weeks) found the higher threshold to be non-inferior, with comparable respiratory and clinical outcomes. The use of a 40% threshold significantly reduced surfactant exposure without increasing complications, supporting its role as a safe and cost-effective strategy for resource-limited settings.</p><p><i>Trial registration</i>: CTRI/2023/12/060562.<Table Float="No" ID="Taba"> <tgroup cols="1"> <colspec align="left" colname="c1" colnum="1" /> <tbody> <row> <entry align="left" colname="c1"> <p><b>What is Known:</b></p> <p>• <i>Early surfactant administration improves outcomes in preterm neonates with RDS.</i></p> <p>• <i>International guidelines generally recommend a FiO₂ threshold of 30% for surfactant therapy.</i></p> </entry> </row> <row> <entry align="left" colname="c1"> <p><b>What is New:</b></p> <p>• <i>This is the first randomized controlled trial directly comparing FiO₂ thresholds of 40% versus 30% for surfactant administration.</i></p> <p>• <i>A 40% threshold was non-inferior to 30% in preterm infants (26–32 weeks)&#xa0;without&#xa0;an increase in adverse outcomes, and&#xa0; thus has the potential to support cost effective neonatal care in resource-limited settings and&#xa0;&#xa0;reduce NICU workload. .</i></p> </entry> </row> </tbody> </tgroup> </Table></p>