Poly-alpha-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn(74) in ST8SiaIV and Asn(89) and Asn(219) in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.