Abstract <p>Leloir glycosyltransferases are instrumental in the synthesis of glycoconjugates. Nucleotide sugars as their donor substrates are still considered expensive making preparative enzymatic syntheses economically unattractive. The review highlights the development and advancements of in situ regeneration cycles that utilize nucleotides as byproducts from glycosyltransferase reactions to synthesize respective nucleotide sugars. This approach reduces costs and avoids inhibition of Leloir glycosyltransferases. Regeneration cycles for ten nucleotide sugars are explored emphasizing enzyme cascades from salvage pathways and nucleotide biosynthesis. Additionally, the review highlights advancements involving sucrose synthase for the in situ regeneration of nucleotide sugars from sucrose. Sucrose synthase as the first example of a reversible glycosyltransferase reaction paved the way to establish economic syntheses of glycosylated natural products. Important aspects like enzyme immobilization and protein fusion to optimize processes are discussed. Overall, the review underscores the significance of advanced in situ regeneration cycles for nucleotide sugars for cost-effective access to high-value glycoconjugates.</p> Key points <p>•<i> Enzyme cascades for in situ regeneration of nucleotide sugars</i></p> <p>•<i> Effective cycles for large-scale synthesis of glycoconjugates</i></p> <p>•<i> Regeneration of nucleotide sugars from sucrose by sucrose synthase</i></p> Graphical abstract <p></p>