Background: Phenylketonuria (PKU) is a hereditary metabolic disorder caused by phenylalanine hydroxylase deficiency. Aim: This research aimed to assess the influence of an educational program on upgrading knowledge and practice of mothers regarding the care of their children suffering from phenylketonuria. Design: A quasi-experiment. Sampling: A convenience sample was used. Setting: At the Genetic & Metabolic Outpatient Clinic at Pediatric Zagazig University Hospital. Tools: Two tools; tool I: A structured interview survey, tool II: Reported practice checklist. Results: revealed that nearly one quarter of studied mothers had satisfactory total knowledge score at pretest compared to most of them post intervention. As well as, nearly three quarters of them had incompetent total practices toward caring of their children with PKU before intervention program that reduced to less than one fifth of them post of program implementation. Moreover, A statistically significant correlation detected between total mothers´ knowledge score and their total reported practice at post intervention (p<0.001).  Conclusion: The mothers had a low knowledge and practice levels regarding the care of their children with PKU, as well as the educational program was effective in upgrading mothers' knowledge and practice toward caring of their children. Recommendation: Continuous educational programs for mothers about phenylketonuria are required to increase their awareness and performance toward the care of their children with phenylketonuria.