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Object To analyze the interaction of PNPLA3 rs738409 polymorphism with physical activity frequency on childhood NAFLD (non-alcoholic fatty liver disease).Method 1027 children and adolescents had carried out fatty liver ultrasonography evaluation, genotyping, and questionnaires on physical activity.Results At multivariate logistic regression analysis considering demographic, anthropometric and genetic determinants, the effect of rs738409 on NAFLD differed with physical activity frequency (<1hour/day: OR=2.51, p<0.001;≥1hour/day:OR=1.06, p=0.815).There was a significant interaction between rs738409 and physical activity frequency (p=0.009) on NAFLD.Conclusion To our knowledge, this is the first report for the interaction of PNPLA3 rs738409 polymorphism with physical activity frequency among Chinese children and adolescents.