Neonatal cholestasis disease (NCD) is a complex and easily mis-diagnosed condition. We analyzed microbiota community structure in feces and measured short-chain fatty acids, bile acids (BAs) and liver function of 12 healthy, 13 NCD, and 13 treated infants after diagnosis. Based on 16S rRNA gene amplicon sequencing and gas-chromatographic-mass-spectrometric analysis of secondary BAs, we identified microbial genera and metabolites that associate with abnormal bile secretion. Streptococcus gallolyticus and Parabacteroides distasonis, and Lactobacillus gasseri had higher relative abundance in healthy and NCD infants respectively. Compared to NCD patients, healthy infants had higher LCA, CDCA and GCDCA fecal concentrations. The three microbial species and three secondary bile acids were selected as potential non-invasive combined biomarkers to diagnose NCD. We propose that microbiotametabolite combined biomarkers could be used for diagnosis of NCD, and this may contribute to improved early clinical diagnosis of NCD in the future. Neonatal cholestasis disease (NCD) affects approximately 1 in every 2500 term infants and is infrequently recognized by primary providers in the setting of physiologic jaundice. Cholestasis jaundice is mostly due to biliary atresia and frequently results from non-biliary atresia 1. The etiology of biliary atresia is unclear but is thought to involve bile duct dysmorphogenesis, viral infection, toxins, chronic inflammation, or autoimmune-mediated bile duct injury 2-5. Non-biliary atresia etiology of neonatal cholestasis may involve bacterial sepsis, galactosemia, tyrosinemia, panhypopituitarism, defective BA synthesis, or obstructive gallstones 1. The complex causes for NCD necessitate improved clinical practice guidelines for the care of infants with cholestasis. Hence, joint general recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition are available for evaluation of NCD in infants, which identify: 1) the measurement of total and conjugated (direct) serum bilirubin for babies at 2 weeks of age; 2) physical examination for hepatomegaly, splenomegaly and appearance of illness; 3) direct visualization of stool pigment; 4) intra-operative cholangiogram and histological examination of the duct remnant 1. However, infants with biliary atresia usually appear healthy and grow normally, which may deceive the parent or physician into believing that the jaundice is physiologic or caused by breastfeeding 6. Thus, development of new or additional biomarkers is considered important in order to improve the care of NCD. Among the most studied causes of NCD in recent years are metabolic diseases and disorders of bile transport and BA synthesis. Cumulative evidence suggests that the composition and function of the gut microbiota plays a prominent role in the occurrence of human metabolic diseases, including type II diabetes 7 , obesity 8 , liver disease 9,10 , atherosclerosis 11...