Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5-10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase is a serine/ threonine kinase that affects multiple cellular processes including tight junction function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize tight junction (TJ) proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells (HUVEC) were studied. Cells were treated with lipopolysaccharide (LPS) to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by trans-epithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii, with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor treated pups compared to NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC.