In preclinical studies of fructose-induced NAFLD, endotoxin appears to play an important role. We retrospectively examined samples from three pediatric cohorts (1) to investigate whether endotoxemia is associated with the presence of hepatic steatosis; (2) to evaluate postprandial endotoxin levels in response to fructose beverage in an acute 24-hour feeding challenge, and (3) to determine the change of fasting endotoxin amounts in a 4-week randomized controlled trial comparing fructose to glucose beverages in NAFLD. We found that adolescents with hepatic steatosis had elevated endotoxin levels compared to obese controls and that the endotoxin level correlated with insulin resistance and several inflammatory cytokines. In a 24-hour feeding study, endotoxin levels in NAFLD adolescents increased after fructose beverages (consumed with meals) as compared to healthy children. Similarly, endotoxin was significantly increased after adolescents consumed fructose beverages for 2 weeks and remained high although not significantly at 4 weeks. In conclusion, these data provide support for the concept of low level endotoxemia contributing to pediatric NAFLD and the possible role of fructose in this process. Further studies are needed to determine if manipulation of the microbiome or other methods of endotoxin reduction would be useful as a therapy for pediatric NAFLD.