Although active inflammatory changes in chronic Crohn's disease (CD) can be detected with serum inflammatory markers, these have low specificity and sensitivity. Stool markers of inflammation, such as M2-pyruvate kinase (M2-PK), permit more direct assessment of mucosal inflammation. The aim of this study was to assess levels of M2-PK in children with active CD and to compare to levels in healthy control children. Fecal levels of M2-PK were measured by immunoassay using stored stool samples from children with untreated (active) CD and healthy control children. Correlations between M2-PK levels and disease activity scores and serum inflammatory markers were performed. Comparison was also made between M2PK and a second fecal inflammatory marker, S100A12. Mean fecal M2-PK levels were higher in the 17 patients with active CD than in the 21 healthy controls (p = 0.0007). M2-PK levels did not correlate with disease activity scores or serum inflammatory markers. There was a trend for children with ileocolonic disease to have higher levels of M2-PK in their stool compared to those with colonic disease or isolated ileal disease. Fecal M2PK did not correlate with fecal S100A12 in children with active CD. Fecal M2-PK is increased in children with active CD, indicating that this marker may be a useful non-invasive marker for gut inflammation. Further studies of M2PK are required in additional settings with larger cohorts of children with CD and with comparison to other stool markers.