Impaired intestinal mucosal integrity and immunity are frequently observed in low-birth-weight (LBW) animals, which lead to inadequate growth and high neonatal mortality. However, the mechanisms of intestinal dysfunction in LBW animals are still unclear. Milk fat globule membrane (MFGM), a protein-lipid complex surrounding the fat globules in milk, has many healthful benefits for animals. Therefore, this study was conducted to explore the effect of MFGM supplementation on intestinal injury and inflammation in LBW mouse pups while being challenged with lipopolysaccharide (LPS). C57BL/6J LBW female neonatal mice were fed on breast milk and divided into four groups, including two normal diet groups (ND; CON group and LPS group) and the diet supplemented with two dosages of MFGM, namely, MFGM100 (ND plus MFGM at 100 mg/kg BW) and MFGM200 (ND plus MFGM at 200 mg/kg BW) from postnatal day (PND) 4 to PND 21. At PND21, pups from the LPS group, MFGM100 group, and MFGM200 group were injected intraperitoneally with LPS while the pups from the CON group were injected with equivalent volume of sterile saline. After 4 h of LPS administration, all pups were slaughtered and then the plasma, mid-ileum, and mid-colon tissue samples were collected. Our results showed that MFGM supplementation promoted the body weight from PND16 to PND21 and attenuated intestinal inflammation manifested by reduced histological damage, decreased secretion of TNF-α, IL-6, IFN-γ, and IL-1β, and improved oxidative stress characterized by increased SOD activity and decreased secretion of MDA. Expression of tight junction proteins (ZO-1, occludin, and claudin-1), MUC1, and MUC2 was increased in MFGM presupplemented groups compared to the LPS-challenged mice with normal diet. Meanwhile, the expression of proinflammatory cytokines and TLRs was decreased by MFGM presupplementation. Collectively, MFGM is a critical nutrient with an ability to improve the growth performance of LBW mouse pups, especially during the LPS challenge, by promoting the intestinal epithelial integrity and inhibiting inflammation through activating of TLR2 and TLR4 signals.