Cingulin (CGN) is a 140 kDa protein, which is localized to the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signaling in cultured cells. To investigate the function of CGN at the organism level, we generated CGN knockout (CGN(-/-)) mice by homologous recombination. CGN(-/-) mice are viable and fertile, and are born at the expected mendelian ratios. Immunohistochemistry, immunofluorescence, electron microscopy and permeability assays of epithelial tissues of CGN(-/-) mice show no cingulin labeling at junctions, a normal localization of TJ proteins, and normal TJ structure and barrier function. Microarray analysis of intestinal cells does not show significant changes in gene expression between CGN(-/-) and CGN(+/+) mice, whereas immunoblotting analysis shows a twofold increase in the levels of claudin-2 protein in the duodenum and the kidney of CGN(-/-) mice, compared to CGN(+/+) littermates. Furthermore, CGN(-/-) mice show an exacerbated response to the ulcerogenic action of cysteamine, whereas acute injury of the colon by dextran sodium sulfate elicits undistinguishable responses in CGN(-/-) and CGN(+/+) mice. We conclude that at the organism level cingulin is dispensable for the structure and barrier function of TJ, and is embedded in signaling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum.