Disruption of tight junctions (TJs) predisposes to bacterial translocation, intestinal inflammation, and necrotizing enterocolitis (NEC). Previously, studies showed that hyaluronan (HA), a glycosaminoglycan in human milk, maintains intestinal permeability, enhances intestinal immunity, and reduces intestinal infections. In this study, we investigated the effects of HA 35 kDa on a NEC-like murine model.Pups were divided into Sham, NEC, NEC+HA 35, and HA 35. Severity of intestinal injury was compared using a modified macroscopic gut scoring and histologic injury grading. The effect of HA 35 on intestinal permeability was determined by measuring FITC dextran and bacterial translocation. RNA and protein expression of TJ proteins (claudin-2, -3, -4, occludin, and ZO-1) were compared between the groups.Pups in the NEC+HA 35 group had increased survival and lower intestinal injury compared to untreated NEC. In addition, HA 35 reduced intestinal permeability, bacterial translocation, and proinflammatory cytokine release. Ileal expression of claudin-2, -3, -4, occludin, and ZO-1 was upregulated in NEC+HA 35 and HA 35 compared to untreated NEC and shams.These findings suggest that HA 35 protects against NEC partly by upregulating intestinal TJs and enhancing intestinal barrier function.