N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST-2) catalyzes the sulfation of mucin-like glycoproteins, which function as ligands for a lymphocyte homing receptor, L-selectin, in the lymph node high endothelial venules (HEVs). We previously showed that GlcNAc6ST-2 is expressed not only in lymph node HEVs but also in the colonic epithelial cells in mice. Here we investigated the regulatory mechanism and physiological significance of colonic expression of GlcNAc6ST-2 in mice. Treatment of a mouse colonic epithelial cell line with butyrate, a short-chain fatty acid produced by anaerobic bacteria, induced GlcNAc6ST-2 expression in the presence of epidermal growth factor. Administration of butyrate in the drinking water stimulated GlcNAc6ST-2 expression in the mouse intestine, indicating that butyrate could serve as a regulatory molecule for the GlcNAc6ST-2 expression in vivo. Immunohistochemical analysis indicated that the sulfation of colonic mucins was greatly diminished in GlcNAc6ST-2-deficient mice. Liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type and GlcNAc6ST-2-deficient mice showed that GlcNAc-6-O-sulfation was the predominant sulfate modification of these mucins, and it was exclusively mediated by GlcNAc6ST-2. After colitis induction by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of wild-type mice, indicating that the sulfation of colonic mucins by GlcNAc6ST-2 has a protective function in experimental colitis. These findings indicate that GlcNAc6ST-2, whose expression is regulated by butyrate, is a major sulfotransferase in the biosynthesis of sulfomucins in the mouse colon, where they serve as a mucosal barrier against colonic inflammation.