Background: Fetal swallowing of amniotic fluid, which contains several cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid might provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. Methods: We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (rHGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC-6 cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Results: Amniotic fluid supplementation of oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by rHGF and blocked by anti-HGF antibody or a PI3K inhibitor. rHGF transactivated several RTKs in IEC-6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, rHGF also reduced the frequency and severity of NEC-like injury in rat pups. Conclusions: Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.