Neonatal cholestasis is the consequence of reduced bile synthesis by the hepatocyte or blocked  bile flow via the intra- or extrahepatic biliary tree, which leads in the buildup of biliary chemicals in the liver, blood, and extrahepatic tissue. Cholestasis inflicts damage on the patients' gut in addition to their liver, irrespective of the etiology. The sole known physiological regulator of intercellular tight junctions is zonulin, which has been shown to be correlated with intestinal permeability. Consequently, it might be used as a metric for compromised gut barrier function.  The aim of this work is to determine whether gut barrier integrity was impaired in infants with biliary atresia in comparison to other cases of cholestasis by measuring serum level of zonulin and intestinal fatty acid binding protein IFABP.  This was a case control study. The study population was composed of 30 infants diagnosed as biliary atresia (BA), 27 infants with cholestasis other than BA and 29 apparently healthy infants serving as a control group. During a two-year period, all patients were recruited from the outpatient clinic and the inpatient ward of the pediatric Hepatology, Gastroenterology, and Nutrition department at the National Liver Institute, Menoufia University, in accordance with the inclusion and exclusion criteria (2022-2023).  They were matched based on their gender and age. Infants with cholestasis had substantially increased serum zonulin levels and IFAB as compared to healthy controls (p < 0.001). Moreover, the serum levels of zonulin were not significantly higher in BA than in cholestasis or any other condition.  Serum zonulin and IFABP concentrations were significantly amplified in cholestasis than healthy control as an indicator of intestinal permeability affection in cholestasis.