The alpha-2,3-sialyltransferase from Neisseria gonorrheae was overproduced in E. coli for exploitation of its substrate specificity and synthetic utility. Several potential acceptor substrates were synthesized in this study, including mono- and oligosaccharides, glycolipids, and glycopeptides and their sulfate derivatives. Some CMP-sialic acid derivatives with modification at the C-5 position were also prepared for evaluation as donor substrates. It was found that the enzyme exhibits a broader acceptor substrate specificity when compared to other sialyltransferases, though the donor specificity is quite limited. Application of the enzyme to the preparative synthesis of representative sialyl glycoconjugates has been demonstrated. On the basis of this work and the work of others, this enzyme is the most versatile and synthetically useful among all sialyltransferases known to date, especially for the synthesis of sulfate-containing glycoconjugates.