Glycosylation is an important post-translational modification catalyzed by glycosyltransferases (GTs), which comprise a large group of enzymes with diversified specificity. Most GTs show high specificity toward donor and acceptor molecules, but some can recognize several donor or acceptor molecules and/or create novel glycosidic linkage types. This promiscuity has profound implications for cellular processes, influencing signaling pathways, protein stability and disease progression. GT promiscuity may result from the structural peculiarities of the enzyme and acceptor, modulation by a ligand, oligomerization with other enzymes, or its subcellular localization. In this review, we discuss current insights into GT promiscuity, highlighting its biological significance and potential associations with human diseases. A better understanding of how GTs work will be essential for developing novel therapeutic approaches and obtaining enzymes with improved properties, which are desirable in the emerging field of glycobiotechnology.