Mucin-type O-glycans, primarily core 1- and core 3-derived O-glycans (O-glycans), are the major mucus barrier components throughout the gastrointestinal tract. Previous reports identified the biological role of O-glycans in the stomach and colon. However, the biological function of O-glycans in the small intestine remains unknown. Using mice lacking intestinal core 1- and core 3-derived O-glycans (IEC C1galt1^-/-;C3GnT^-/- or DKO), we found that DKO mice developed spontaneous duodenal tumors around 1 year of age. Tumor incidence did not increase with age; however, tumors advanced in aggressiveness by 20 months. Duodenal tumor development and O-glycan deficiency was associated with reduced luminal mucus in DKO mice prior to and within tumors. Altered intestinal epithelial homeostasis with enhanced baseline crypt proliferation characterizes these phenotypes as assayed by Ki67 staining. In addition, FISH analysis reveals a significantly lower bacterial burden in the duodenum compared to the large intestine. This phenotype is not reduced with antibiotic treatment, implying O-glycosylation defects, rather than bacterial-induced inflammation, causes spontaneous duodenal tumorigenesis. Moreover, inflammatory responses in DKO duodenal mucosa are mild as assayed with histology, qPCR for inflammation-associated cytokines, and immunostaining for immune cells. Importantly, inducible deletion of intestinal O-glycans in adult mice leads to analogous spontaneous duodenal tumors, though with higher incidence and heightened severity compared to mice with O-glycans constitutive deletion. In conclusion, these studies reveal O-glycans within the small intestine are critical determinants of duodenal cancer risk.