Aberrant glycosylation has been observed in many types of cancer, but the mechanism of glycosylation change is still poorly understood. To elucidate relationships between glycosylation and colon cancer progression, we analyzed glycosylation status of beta-haptoglobin (beta-Hp) obtained from 46 cancer patients, 14 inflammatory bowel disease patients and 38 normal subjects. Aleuria aurantia lectin reactivity with cancer beta-Hp was much higher than in the other two study groups. These results were confirmed by lectin blotting and microarray assay using other lectins directed to fucosyl residues. Levels of such glycans were correlated with stage of colon cancer progression. Reactivity with fucosylated glycans was eliminated by treatment with a1-3/4 fucosidase but not a1-6 fucosidase, indicating that enhanced lectin reactivity with the fucose moiety of colon cancer beta-Hp is due to Fuca1-3/4GlcNAc. Moreover, site-specific glycan occupancy was determined by sequential LC/MS analysis. Mass spectrometric analysis showed that fucosylation of beta-Hp was higher in colon cancer patients than in other subjects. In particular, fucosylation at Asn 241 of beta-Hp in sera of colon cancer patients was clearly higher than in the other groups, and the ratio of fucosylated glycopeptides containing Asn 241 decreased greatly after treatment with a1-3/4 fucosidase. In conclusion, the level of a1-3/4 fucosyl epitope at Asn 241 of beta-Hp is potentially useful as a novel marker for colon cancer.