Necrotizing enterocolitis (NEC) remains a major challenge in neonatology. Little is known about NEC pathophysiology apart from the presence of pre-event gut dysbiosis. Here, we applied broad range metabolomics to stools obtained 1–5 days before NEC developed from 9 cases (9 samples) and 19 (32 samples) controls matched for gestational age at birth and birth weight. The 764 identified metabolites identified six pathways that differ between cases and controls. We pursued sphingolipid metabolism because cases had decreased ceramides and increased sphingomyelins compared to controls, and because of the relevance of sphingolipids to human inflammatory disorders. Targeted analysis of samples from 23 cases and 46 controls confirmed the initial broad range observations. While metabolites provided only 73% accuracy of classification by machine learning, hierarchical clustering defined a sphingolipid associated grouping that contained 60% of the cases but only 13% of the controls, possibly identifying a pathophysiologically distinct subset of NEC. The clustering did not associate with any of the analyzed clinical and sample variables. We conclude that there are significant changes in sphingolipid metabolism components in pre-NEC stools compared to controls, but our data urge circumspection before using sphingolipids as broadly applicable predictive biomarkers.