The carbohydrate determinants Sd^a and sialyl Lewis x (sLex) both result from substitution of an α2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) β1,4-linked to Gal and the second by an α1,3-linked fucose on N-acetylglucosamine. The Sd^a antigen is synthesized by Sd^a β1,4-N-acetylgalactosaminyltransferase II (β4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between β4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether β4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. β4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd^a antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd^a antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, β4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd^a and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of α1,3-FucTs on 3′-sialyllactosamine parallels that of β4GalNAcT-II; and (iii) both β4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription–polymerase chain reaction analysis reveals that the transcripts of β4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3′-sialyllactosamine acceptors and that β4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.