Background: Down syndrome is a chromosomal defect causing intellectual impairment and physical abnormalities. A healthy child is born with 46 chromosomes, whereas a child with Down syndrome has an extra copy of chromosome 21. Aim of the study: This study aimed to assess mothers' knowledge and practices toward care of their children with Down syndrome. Research design: A descriptive research design was used. Research setting: The study was conducted in Pediatric Outpatient Genetic Clinic at Specialized Pediatric Hospital which is affiliated to Secretariat of Specialized Medical Center at Benha City. Study subjects: A purposive sample of 55 mothers accompanying their children. Tools of data collection: Two tools were used; Tool I: A structured interview questionnaire sheet: It consisted of five parts; Part 1: Personal characteristics of the studied mothers, Part 2: Characteristics of the studied children, Part 3: Medical history of the studied children, Part 4: Previous obstetric history of the studied mothers, Part 5: Mothers' knowledge about Down syndrome. Tool II: Mothers' reported practices toward care of their children with Down syndrome. Results: The majority of the studied mothers had unsatisfactory level of knowledge regarding Down syndrome, meanwhile more than two thirds of them had inadequate level of total reported practices regarding caring of their children with down syndrome. Conclusion: There was a highly statistical positive correlation between total level of the mothers' knowledge and their total reported practices level toward care of their children with Down syndrome. Recommendations: Designing and implementing an educational program for mothers to improve their knowledge and practices about care for their children with Down syndrome.