Colonic paracellular permeability is regulated by various factors, including dynamics of the cytoskeleton. Recently, ACF7 has been found to play a critical role in cytoskeletal dynamics as an essential integrator. To elucidate the physiological importance of ACF7 and paracellular permeability, we conditionally knocked out ACF7 in the intestinal mucosa of mice. Histopathological findings indicated that ACF7 deficiency resulted in significant interstitial proliferation and columnar epithelial cell rearrangement. Decreased colonic paracellular permeability was detected using a Ussing chamber and the FITC-inulin method. In order to clarify the underlying mechanism, we further analyzed the expression levels of three important tight junction proteins. Downregulation of ZO-1, occludin and claudin-1 was identified. Immunofluorescence provided strong evidence that ZO-1, occludin and claudin-1 were weakly stained. We hypothesized that ACF7 regulates cytoskeleton dynamics to alter mucosal epithelial arrangement and colonic paracellular permeability.