One of the major challenges for neonatal care is prematurity and related problems, particularly respiratory distress syndrome (RDS). There is a link between lung development and vitamin D has been shown in numerous studies. The aim of the study was: To evaluate the effects of vitamin D supplementation guidelines on nurses' care and associated outcomes for preterm neonates with respiratory distress syndrome. Subjects and Methods: Design: A quasi-experimental research design was used to achieve the study's aim.  Setting: The study was conducted at the neonatal intensive care unit (NICU) affiliated to Sohag University Hospital. Sample: The study included a convenience sample of (50) nurses working in the previously mentioned study setting with a purposive sample included 50 preterm neonates chosen from the previously mentioned setting with respiratory distress syndrome receiving traditional therapy plus vitamin D 400 IU/day. Tools: Tool (1) A structured interviewing schedule; Tool (II): an Observation Checklist to assess the nurses' practice regarding their care provided for the neonates with respiratory distress syndrome; Tool (III): Preterm neonatal assessment sheet. Results: The majority of nurses had average knowledge after the implementation of the instructional guidelines. Also, preterm neonates supplemented with vitamin D had a significant improvement in all pulmonary function parameters, lower Downs score, and less hospital stays. Conclusion: Vitamin D supplementation guidelines have positive effects on improving nurses' knowledge and practices regarding respiratory distress syndrome and Vitamin D supplementation. Also, improved associated outcomes for preterm neonates with respiratory distress syndrome such as decreases the severity, rate of complications, and duration of hospital stay in the preterm neonates with respiratory distress syndrome who received vitamin D/Day post guidelines implementation compared to pre guidelines implementation. Recommendations: Providing nurses with well-planned instructional guidelines to improve their knowledge and practices regarding Vitamin D supplementation.  Vitamin D should be supplemented to preterm neonates with respiratory distress syndrome