The actin cytoskeleton is a critical regulator of intestinal mucosal barrier permeability, and the integrity of epithelial adherens junctions (AJ) and tight junctions (TJ). Non muscle myosin II (NM II) is a key cytoskeletal motor that controls actin filament architecture and dynamics. While NM II has been implicated in the regulation of epithelial junctions in vitro, little is known about its roles in the intestinal mucosa in vivo. In this study, we generated a mouse model with an intestinal epithelial-specific knockout of NM IIA heavy chain (NM IIA cKO) and examined the structure and function of normal gut barrier, and the development of experimental colitis in these animals. Unchallenged NM IIA cKO mice showed increased intestinal permeability and altered expression/localization of several AJ/TJ proteins. They did not develop spontaneous colitis, but demonstrated signs of a low-scale mucosal inflammation manifested by prolapses, lymphoid aggregates, increased cytokine expression, and neutrophil infiltration in the gut. NM IIA cKO animals were characterized by a more severe disruption of the gut barrier and exaggerated mucosal injury during experimentally-induced colitis. Our study provides the first evidence that NM IIA plays important roles in establishing normal intestinal barrier, and protection from mucosal inflammation in vivo.