The objective of this study was to evaluate the effect of a self-learning package on mothers’ knowledge and practices towards caring for their children with phenylketonuria. A pre/post quasi-experimental study was conducted, including 128 mothers of children diagnosed with phenylketonuria. A specifically designed and validated questionnaire was used to evaluate mothers’ knowledge and reported practices toward their children before and after participating in the educational program. There was a highly positive association between knowledge and reported practice (.674 and .398). The self-learning package had a positive impact on mothers’ knowledge and practices. Consequently, educational programs should be provided to all mothers of newly diagnosed cases to improve their children’s adherence to the therapeutic regimen.