The mouse intestinal mucus is mainly made up by the gel-forming Muc2 mucin and the stomach surface mucus Muc5ac, both extensively O-glycosylated. The oligosaccharide diversity provides a vast library of potential recognition sites for both commensal and pathogenic organisms. The mucin glycans are thus likely very important for the selection and maintenance of a stable intestinal flora. Here we have explored the O-glycan patterns of the mouse gastrointestinal tract mucins. The mucins from the mucus of the distal and proximal colon, ileum, jejunum, duodenum, and stomach of conventionally raised wild-type (C57BL/6) mice were separated by composite gel electrophoresis. The O-linked glycans were released by reductive elimination and structurally characterized by liquid chromatography-mass spectrometry. The mucins glycans were mostly core 2 type [Galβ1–3(GlcNAcβ1–6)GalNAcol], but also core 1 (Galβ1–3GalNAcol). In the stomach about half of the Muc5ac mucin O-glycans were neutral and many monosulfated, but with a low grade of sialylation and fucosylation. Mouse ileum, jejunum, and duodenum had similar glycan patterns dominated by sialylated and sulfated core 2 glycans, but few fucosylated. Colon was on the other hand dominated by highly charged fucosylated glycans. The distal colon is different from the proximal colon because different biosynthetic pathways are utilized, although sialylated and sulfated glycans were highly abundant in both parts. The sulfation was higher in the distal colon, whereas sialic acid was more common in the proximal colon. Many fucosylated glycans were found in both the proximal and distal colon. Thus the mucin O-glycans vary along the mouse gastrointestinal tract.