The highly sulfated polysaccharide sea cucumber chondroitin sulfate (SCCS) can alleviate intestinal damage and display strong anti-food-allergic activity. The O-glycopattern levels in colonic mucin are closely related to the its protective effect on function of the intestinal barrier. However, the effect of the SCCS on colonic mucin O-glycan has not been investigated. In this study, ovalbumin (OVA)-sensitized allergic mice and SCCS treatment were used. Mouse colonic mucin O-glycome was released and analyzed through reductive β-elimination combined with PGC-LC-MS. A total of presumptive 20 neutral and 28 acidic O-glycan structures were identified, in which the core 2 type acidic O-glycan structure is predominant in Balb/c female mice. Treatment with OVA and SCCS did not change the numbers of colon mucin O-glycan type, but the expression level of total O-glycosylation was more abundant in the SCCS group mice than in the OVA group (1.8-fold), especially for acidic O-glycans (co-modified by fucose and sulfate groups). Furthermore, supplementation with SCCS reversed most of the O-glycan decreasing trend, which may be associated with a return to healthy levels of gut microbiota. In conclusion, our results demonstrate that SCCS could restore colonic mucin O-glycosylation levels and intestinal homeostasis and contribute to enhancing intestinal barrier function.