Background: The stimulating effect of surgery may be explained by the facts that stress hormones released in response to pain target. Activation of the stress response the endocrine response is activated by afferent neuronal impulses from the site of injury and metabolic changes induced by the stress response are satisfied and to provide an alternate source of metabolic fuel for tissues. Aim of the study: Wasto evaluate the effect of educational program for nurses regarding neonatal hormonal changes and metabolic stress response after surgery. Design: Quasi experimental study design was used. Setting: This study was conducted at Surgical Neonatal Intensive Care Units at Specialized Children Hospital in Benha City. Sample: A convenient sample of 60 nurses and a purposive sample of 50 neonates. Tools of data collection: Four tools were used for data collection (Characteristics of children and nurses structured interviewing questionnaire, behavioral observation checklist for neonates, observational checklist to assess nurses’ level practice and pain assessment scale). Results: Majority (93%) studied nurses had competent level of knowledge score in post-educational program, while the majority (90.3%) of them had good score of practice post-educational program intervention.Conclusion: Educational program was effective in improving nurses’ knowledge and practice after implementation of educational program and more than three quarters (78%) of studied neonates had moderate pain in post intervention. Recommendation: Educational program should be integrated into pediatric nursing curricula to enhance knowledge and skills regarding hormonal changes and metabolic stress response after surgery.