Background: Necrotizing enterocolitis (NEC) is a disease with prevalent and serious intestinal inflammation that poses a significant threat to the lives of newborns. Human milk has been shown to prevent and treat the occurrence of NEC; however, the underlying mechanisms remain unclear. MAMP-1 is a significantly overexpressed endogenous peptide derived from beta-casein extracted from the human milk of premature mothers, which is resistant to gastrointestinal conditions and exhibits favorable physicochemical properties. This study aims to investigate the mechanism by which the human milk-derived peptide MAMP-1 mitigates NEC in mice, offering new insights for clinical treatment. Methods: The C57BL/6 mice were categorized into three distinct groups randomly on the 7th day after birth, with 40 mice in each group. The NEC model was established using "artificial feeding + hypoxia + cold", and the MAMP-1 group received daily MAMP-1 gavage during NEC modeling. Ileum tissues and feces were collected. Pathological damage in the intestines was evaluated by H&E staining. ZO-1 expression was analyzed through immunofluorescence staining and western blot. Apoptosis in the intestine was assessed using western blot and TUNEL staining. The effects of signaling pathways were confirmed through western blot and RNA sequencing. The expression of inflammatory factors was assessed using RT-PCR. 16S high-throughput sequencing was used to determine the diversity and abundance of the gut microbiota. Results: MAMP-1 reduced the mortality rate of NEC mice, alleviated ileum injury, increased the ZO-1 expression of the intestinal barrier, reduced the apoptotic protein expression, lowered the TUNEL positive area, increased anti-apoptotic protein expression, and reduced the levels of TLR4, P-PI3K, P-AKT, and NF-kappa B, leading to a reduction in the release of inflammatory cytokines. Furthermore, MAMP-1 decreased the abundance of harmful bacteria and increased the abundance of beneficial bacteria at both the phylum and genus levels. Conclusion: MAMP-1 might inhibit the TLR4-PI3K-AKT-NF-kappa B signaling pathways, further reducing inflammation factor release, and might decrease intestinal cell apoptosis. Results indicated that MAMP-1 might alleviate intestinal damage in NEC mice. Meanwhile, MAMP-1 might positively modulate the composition of the microbiota of NEC mice and further achieve the preventive and therapeutic effect of MAMP-1 on NEC.