Background Phenylketonuria is an inherited metabolic disease caused by phenylalanine hydroxylase deficiency. Aim This study aimed to assess mothers' knowledge and practice regarding care of their children suffering from Phenylketonuria. Design A descriptive design was utilized in this study. Sampling A purposive sample comprised of 91 mothers having children with phenylketonuria was selected to conduct this study. Setting This study was conducted at specialized genetic outpatient clinic in children's hospital affiliated to Ain Shams University Hospital. Tools Two tools were used in this study Tool (1) A structured questionnaire to assess mothers' knowledge regarding Phenylketonuria. Tool (2) Practice checklists to assess mothers' reported practice regarding care of their children with phenylketonuria. Results Findings of this study showed that, the total knowledge of mothers was unsatisfactory, the total practice was incomplete and there was a highly statistically significant relation between total knowledge and practice of mothers and their characteristics particularly educational level and occupation. Conclusion The studied mothers had low level of knowledge and practice regarding care of their children with Phenylketonuria, there was a statistically significant difference between studied mothers' total level of knowledge and reported practice and their characteristics. Recommendation Continuous educational programs for mothers about phenylketonuria are required to increase their awareness and performance regarding care of their children with phenylketonuria.