Surveillance endoscopy with biopsy is the standard method to monitor intestinal transplant recipients but it is invasive, costly and prone to sampling error. Early noninvasive biomarkers of intestinal rejection are needed. In this pilot study we applied metabolomics to characterize the metabolomic profile of intestinal allograft rejection. Fifty-six samples of ileostomy fluid or stool from 11 rejection and 45 nonrejection episodes were analyzed by ultraperformance liquid chromatography in conjunction with Quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). The data were acquired in duplicate for each sample in positive ionization mode and preprocessed using XCMS (Scripps) followed by multivariate data analysis. We detected a total of 2541 metabolites in the positive ionization mode (mass 50–850 Daltons). A significant interclass separation was found between rejection and nonrejection. The proinflammatory mediator leukotriene E4 was the metabolite with the highest fold change in the rejection group compared to nonrejection. Water-soluble vitamins B2, B5, B6, and taurocholate were also detected with high fold change in rejection. The metabolomic profile of rejection was more heterogeneous than nonrejection. Although larger studies are needed, metabolomics appears to be a promising tool to characterize the pathophysiologic mechanisms involved in intestinal allograft rejection and potentially to identify noninvasive biomarkers.