Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Maternal milk has been suggested to be partially protective against NEC; however, the mechanisms of this protection are not defined. The aim of this study was to examine the effect(s) of artificial feeding of rat milk (RM)-versus cow milk-based rat milk substitute (RMS) on the development of NEC in a neonatal rat model and elucidate the role of inflammatory cytokines in NEC pathogenesis. Newborn rats were artificially fed with either collected RM or RMS. Experimental NEC was induced by exposure to asphyxia and cold stress and evaluated by histologic scoring of damage in ileum. Intestinal cytokine mRNA expression was determined by real-time PCR. Cytokine histologic localization was performed by confocal microscopy. Similar to human NEC, artificial feeding of RM reduces the incidence and severity of NEC injury in neonatal rats. Freezing and thawing of collected RM did not eliminate the protective effect of maternal milk. Ileal IL-10 expression was significantly increased in the RM group compared with RMS. Increased IL-10 peptide production was detected in the RM group with signal localized predominantly in the cytoplasm of villus epithelial cells. These results suggest that the protective effect of maternal milk is associated with increased production of anti-inflammatory IL-10 in the site of injury. Better understanding of the mechanisms underlying these protective effects could be beneficial either in the prevention of NEC or in the development of future therapeutic strategies to cure NEC.