Background: Iron deficiency anemia is a global public health problem; the pregnant women are the most vulnerable groups to iron deficiency anemia during pregnancy causing maternal deaths. The aim of this research: Was to evaluate the effect of nutritional guideline on knowledge, attitude and practices of pregnant women regarding prevention of iron deficiency anemia. Design: A quasi-experimental design (one group pre- posttest). Sample: A convenient sample of 90 pregnant women were included in the present study. Setting: The present study was conducted in outpatient clinics of obstetrics and gynecological department in Benha university hospitals. Tools of data collection: Three tools were used: Tool I: A structured interviewing questionnaire. Tool II: Modified likert scale. Tool III: Assessment of practices sheet for preventive iron deficiency anemia.  Results: The result of the present study revealed that there was a highly statistically significant difference in relation to total studied women' knowledge, attitude and practices about prevention of iron deficiency anemia pre and post phases of nutritional guideline implementation. Moreover, there was a positive statistically significant correlation between total knowledge, total attitude and total practice scores pre and post phases of nutritional guideline implementation. Conclusion: The nutritional guideline had a positive effect on improving knowledge, attitude and practices of pregnant women regarding prevention of iron deficiency anemia. Recommendations: Counseling and health education programs should be applied on pregnant women to improve knowledge, attitude and practices regarding prevention of iron deficiency anemia during pregnancy.