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Abstract The integrity of the intestinal mucus barrier is crucial for human health, as it serves as the body's first line of defense against pathogens. However, postnatal development of the mucus barrier and interactions between maturity and its ability to adapt to external challenges in neonatal infants remain unclear. In this study, we unveil a distinct developmental trajectory of the mucus barrier in preterm piglets, leading to enhanced mucus microstructure and reduced mucus diffusivity compared to term piglets. Notably, we found that necrotizing enterocolitis (NEC) is associated with increased mucus diffusivity of our large pathogen model compound, establishing a direct link between the NEC condition and the mucus barrier. Furthermore, we observed that addition of sodium decanoate had varying effects on mucus diffusivity depending on maturity and health state of the piglets. These findings demonstrate that regulatory mechanisms governing the neonatal mucosal barrier are highly complex and are influenced by age, maturity, and health conditions. Therefore, our results highlight the need for specific therapeutic strategies tailored to each neonatal period to ensure optimal gut health.