Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional alpha2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcbeta1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward beta-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes.