ABSTRACT Objective Adverse physiology and antibiotic exposure devastate the intestinal microbiome in critical illness. Time and cost implications limit the immediate clinical potential of microbial sequencing to identify or treat intestinal dysbiosis. Here, we examined whether metabolic profiling is a feasible method of monitoring intestinal dysbiosis in critically ill children. Design Prospective multicenter cohort study. Setting Three UK-based pediatric intensive care units. Patients Mechanically ventilated critically ill (n=60) and age-matched healthy children (n=55). Interventions Collection of urine and fecal samples in children admitted to the PICU. A single fecal and urine sample was collected in healthy controls. Measurements Untargeted and targeted metabolic profiling using 1H-Nuclear Magnetic Resonance (1H-NMR) Spectroscopy and Liquid Chromatography-Mass Spectrometry (LC-MS) or urine and fecal samples. This was integrated with analysis of fecal bacterial 16S rRNA profiles and clinical disease severity indicators. Main Results We observed separation of global urinary and fecal metabolic profiles in critically ill compared to healthy children. Urinary excretion of mammalian-microbial co-metabolites hippurate, 4-cresol sulphate and formate were reduced in critical illness compared to healthy children. Reduced fecal excretion of short chain fatty acids (including butyrate, propionate and acetate) were observed in the patient cohort, demonstrating that these metabolites also distinguished between critical illness and health. Dysregulation of intestinal bile metabolism was evidenced by increased primary and reduced secondary fecal bile acid excretion. Fecal butyrate correlated with days free of intensive care at 30 days (r=0.38, p=0.03), whilst urinary formate correlated inversely with vasopressor requirement (r=-0.2, p=0.037). Conclusions Disruption to the functional activity of the intestinal microbiome may result in worsening organ failure in the critically ill child. Profiling of bacterial metabolites in fecal and urine samples may support identification and treatment of intestinal dysbiosis in critical illness.