INTRODUCTION: Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease. Its onset might be triggered by Toll-Like Receptor 4 (TLR4) activation via bacterial lipopolysaccharide (LPS). We hypothesize that a deficiency of intestinal alkaline phosphatase (IAP), an enzyme secreted by enterocytes that dephosphorylates LPS, may contribute to NEC development. METHODS: In this prospective pilot study, we analyzed intestinal resection specimens from surgical NEC patients, and from patients undergoing Roux-Y reconstruction for hepatobiliary disease as controls. We assessed IAP activity via enzymatic stainings and assays and explored IAP and TLR4 co-localization through immunofluorescence. RESULTS: The study population consisted of five NEC patients (two Bell's stage IIb and three-stage IIIb, median (IQR) gestational age 25 (24-28) weeks, postmenstrual age at diagnosis 28 (26-31) weeks) and 11 controls (unknown age). There was significantly lower IAP staining in NEC resection specimens [49 (41-50) U/g of protein] compared to controls [115 (76-144),  = 0.03]. LPS-dephosphorylating activity was also lower in NEC patients [0.06 (0-0.1)] than in controls [0.3 (0.2-0.5),  = 0.003]. Furthermore, we observed colocalization of IAP and TLR4 in NEC resection specimens. CONCLUSION: This study suggests a significantly lower IAP level in resection specimens of NEC patients compared to controls. This lower IAP activity suggests a potential role of IAP as a protective agent in the gut, which needs further confirmation in larger cohorts.