Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared with their wild-type counterparts, nmMLCK mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels after burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of fluorescein isothiocyanate-dextran (4 kd), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP (N-formyl-methionyl-leucyl-phenylalanine)-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.