Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O–sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer–specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2–based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2–based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1– and core 2–based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3−)β1-3Galβ1–4GlcNAc(6-SO3−)β1–3Galβ1–3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.