In vertebrates, sialylated glycans participate in a wide range of biological processes and affect the development and function of the nervous system. While the complexity of glycosylation and the functional redundancy among sialyltransferases provide obstacles for revealing biological roles of sialylation in mammals,Drosophilapossesses a sole vertebrate-type sialyltransferase,Drosophilasialyltransferase (DSiaT), with significant homology to its mammalian counterparts, suggesting thatDrosophilacould be a suitable model to investigate the function of sialylation. To explore this possibility and investigate the role of sialylation inDrosophila, we inactivated DSiaTin vivoby gene targeting and analyzed phenotypes ofDSiaTmutants using a combination of behavioral, immunolabeling, electrophysiological, and pharmacological approaches. Our experiments demonstrated that DSiaT expression is restricted to a subset of CNS neurons throughout development. We found thatDSiaTmutations result in significantly decreased life span, locomotor abnormalities, temperature-sensitive paralysis, and defects of neuromuscular junctions. Our results indicate that DSiaT regulates neuronal excitability and affects the function of a voltage-gated sodium channel. Finally, we showed that sialyltransferase activity is required for DSiaT functionin vivo, which suggests thatDSiaTmutant phenotypes result from a defect in sialylation of N-glycans. This work provided the first evidence that sialylation has an important biological function in protostomes, while also revealing a novel, nervous system-specific function of α2,6-sialylation. Thus, our data shed light on one of the most ancient functions of sialic acids in metazoan organisms and suggest a possibility that this function is evolutionarily conserved between flies and mammals.