Background: The Arterio-Venous Fistula (AVF) is the most common vascular access used in hemodialysis because AVF   provides good blood flow for dialysis lasts longer than other types of access and is less likely to get infected or cause blood clots than other types of access. Educational guidelines are important for nurses working in pediatric hemodialysis units to improve children outcomes and reduce healthcare costs. Aim: This study aimed to evaluate the effect of educational guidelines on nurses' performance related to care of AVF puncture for children undergoing hemodialysis. Research design: A quasi-experimental design was utilized in this study. Setting: This study was conducted at pediatric hemodialysis unites at Benha and Menufiya University Hospitals. Subjects:A convenience sampling of 41 nurses working in the previous mentioned settings and a convenience sampling of 46 children.Tools of data collection: Two tools were used, I: Structured interviewing questionnaire and II: Observational checklists. Results: Most of the studied nurses had good level of knowledge and most of them had satisfactory level of practices after educational guidelines implementation. Conclusion: The educational guidelines was effective in improving nurses' knowledge and practices level related to care of arteriovenous fistula for children undergoing hemodialysis. Recommendations: Provision of continuous education guidelines in order to update nurses' knowledge and practices.