Background: Developmentally supportive care is an integrated nursing practice that ensuring that the neonatal intensive care unit environment is as similar to the intrauterine environment as possible to maintain the physiological stability and optimal growth and development of preterm infants. Aim: the study aims to evaluate the impact of instructural program about developmental supportive care for enhancing pediatric nurses’ performance in neonatal intensive care units. Subjects and Method: Design: A quasi experimental design was used, with one group employed to conduct the study. Setting: The study was conducted in neonatal intensive care units at Kafr Saad hospital and El- Azhar university hospital, Egypt. Subjects: A purposive sample consisting of all available nurses working at neonatal intensive care units at the above-mentioned study settings (50), Tools: A structured questionnaire, divided into three parts; personal data of studied nurses, assessment of pediatric nurses’ knowledge regarding developmental supportive care in NICU and, observational checklist of nurses’ practices about the developmental supportive care for neonates. Results: All nurses exhibited unsatisfactory knowledge levels Preprogram; while a significant improvement was observed post program implementation. Regarding total practice of developmental supportive care; a significant increase from 0% pre-program to 36% at the follow-up stage was observed in positioning, as well as for total practice of massage and touch; there was a significant increase from 0% pre-program to 76% post-program and 20% at follow-up. As for total practices of nurses regarding developmental supportive care in the neonatal intensive care unit, there was a significant increase in adequate practice from 0% pre-program to 8% post-program. Conclusion: The training program about developmental supportive care was effective in improving nurses’ performance. Recommendation: Continuous training about developmental supportive care for nurses working in neonatal intensive care units is recommended.