Core 1- and 3-derived mucin-type O-glycans are primary components of the mucus layer in the colon. Reduced mucus thickness and impaired O-glycosylation are observed in human ulcerative colitis. However, how both types of O-glycans maintain mucus barrier function in the colon is unclear. We found that C1galt1 expression, which synthesizes core 1 O-glycans, was detected throughout the colon, whereas C3GnT, which controls core 3 O-glycan formation, was most highly expressed in the proximal colon. Consistent with this, mice lacking intestinal core 1-derived O-glycans (IEC C1galt1^-/-) developed spontaneous colitis primarily in the distal colon, whereas mice lacking both intestinal core 1- and 3-derived O-glycans (DKO) developed spontaneous colitis in both the distal and proximal colon. DKO mice showed an early onset and more severe colitis than IEC C1galt1^-/- mice. Antibiotic treatment restored the mucus layer and attenuated colitis in DKO mice. Mucins from DKO mice were more susceptible to proteolysis than wild-type mucins. This study indicates that core 1- and 3-derived O-glycans collectively contribute to the mucus barrier by protecting it from bacterial protease degradation and suggests new therapeutic targets to promote mucus barrier function in colitis patients.