Breastfeeding is an important practice in Egypt from the first day of childbirth by giving colostrum milk. Colostrum is contained large amounts of IgA antibodies that promoting health, reducing diarrheal disease among neonates, and providing excellent immune support. Aim: To evaluate the effect of instructional guidelines on pregnant women's knowledge, attitude, and practices regarding colostrum feeding. Design: Quasi-experimental research design was conducted in this study. Setting: The current study was conducted at the Antenatal Outpatient Clinic at South Valley University Hospital. Subjects: A purposive sample included 100 pregnant womenwere enrolled in the study. One tool was used, a structured interview questionnaire composed of: part I Sociodemographic characteristics: part II: Pregnant women's knowledge assessment sheet about colostrum feeding, part III: Pregnant women's attitude towards colostrum feeding and part IV: Pregnant women's practices of colostrum feeding. Results: Most women did not have knowledge about the importance of colostrum and unsatisfactory level of knowledge toward colostrum feeding was reported, negative attitudes and inadequate practices among pregnant women for colostrum feeding were presented by the studied women pre instructional guidelines implementation. Meanwhile, knowledge, attitude, and practices of the pregnant women had improved post one month of the guidelines implementation. Conclusion: Instructional guidelines were effective in improving pregnant women's knowledge, attitude, and practices regarding colostrum feeding. Recommendations: Educational programs about colostrum feeding should be taught and discussed in the antenatal care follow-up visits to all mothers.