Objective To determine whether longitudinal measurements of fecal S100A12, a fecal marker of intestinal inflammation, can identify very low birth weight infants at risk for necrotizing enterocolitis (NEC). Study design This prospective study included 145 preterm infants with birth weight <1500 g. Meconium and stool samples (n = 843) were collected prospectively on alternate days for 4 weeks, and fecal S100A12 and calprotectin were measured by enzyme-linked immunosorbent assay. Results Eighteen patients (12.4%) developed NEC. Gestational age and birth weight were significantly lower in the patients with NEC compared with unaffected reference infants. Fecal S100A12 levels were significantly higher in patients with severe NEC at onset of disease and also, in contrast to fecal calprotectin, at 4-10 days before onset of NEC compared with unaffected reference infants (ideal cutoff value, 65 μg/kg; sensitivity, 0.76; specificity, 0.56). Conclusions Fecal S100A12 level may be a helpful marker for predicting disease severity and early risk assessment for subsequent development of NEC. However, the use of fecal S100A12 as a predictive biomarker for NEC in very low birth weight infants may be limited due to a high interindividual and intraindividual variability in S100A12 fecal excretion.