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Neonatal necrotizing enterocolitis (NEC) is a serious inflammatory gastrointestinal disease in premature infants. Lactobacillus acidophilus is a probiotic, which exhibits many beneficial effects on the digestive and immune systems. In a rat model of NEC, administration of L. acidophilus CICC6074 reduced NEC symptoms, intestinal apoptosis and the levels of inflammatory cytokines TNF-α and IL-6. LiCl-treated L. acidophilus CICC6074 had less influence on the apoptosis index and TNF-α levels than untreated LA. In a TNF-α-infected Caco-2 cell model, purified L. acidophilus CICC6074 surface-layer protein (SLP) caused lower intestinal epithelial apoptosis, while decreasing IL-6 secretion in both the apical and basolateral compartments. SLP also reduced IL-6 and caspase-3 production in a lipopolysaccharide (LPS)-induced Caco-2 cell model. The results showed that L. acidophilus CICC6074 has the ability to reduce NEC symptoms. SLP of LA may play a role in reducing NEC symptoms by suppressing intestinal cell apoptosis and modulating intestinal immune function.